Fluorescent detector systems for the detection of chemical perturbations in sterile storage devices

ABSTRACT

System and method for detecting and measuring chemical perturbations in a sample. The system and method are useful for non-invasive pH monitoring of blood or blood products sealed in storage bags.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/948,892, filed Jul. 23, 2015 (now U.S. Pat. No. 9,217,170), which isa continuation of U.S. patent application Ser. No. 11/789,431, filedApr. 23, 2007 (now U.S. Pat. No. 8,497,134), which claims the benefit ofU.S. Patent Application No. 60/794,193, filed Apr. 21, 2006; and is acontinuation-in-part of U.S. patent application Ser. No. 11/207,580,filed Aug. 19, 2005 (now U.S. Pat. No. 7,608,460), which claims thebenefit of U.S. Provisional Application No. 60/602,684, filed Aug. 19,2004, and U.S. Provisional Application No. 60/674,393, filed Apr. 22,2005, each of which is expressly incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Platelets are a component of blood comprised of anucleate megakaryocytefragments that circulate in the blood for about 10 days (van der Meer,Pietersz et al. 2001; Dijkstra-Tiekstra 2004). As they age in thecirculatory system platelets are known to undergo biochemical changesthat eventually leads to their clearance in the spleen and liver. Whenseparated as a component of whole blood, platelets are routinelyconcentrated, resuspended in plasma and/or platelet additive solutions,leukoreduced by passage through a filtration device and stored inplatelet storage bags which are kept on flatbed agitators for 5 to 7days at a temperature of 22° C.

The measurement of pH and other parameters during preparation andstorage of blood components are necessary in order to provide a safe andeffective product. For example the storage of platelets at 22° C.requires testing for the presence of microbiological contamination toprevent undesired side effects such as sepsis as a result of infusioninto the patient. Under the American Association of Blood Banks(A.A.B.B.) standard 5.1.5.1, blood banks or transfusion services areinstructed to have methods to limit and detect bacterial contaminationin all platelets. The growth of bacteria in platelet concentrates (PCs)can be monitored by utilizing reagent dipsticks for pH and glucosehowever the time of detection following inoculation with a low dose oforganisms (e.g. 50 colony forming units/mL) varies from organism toorganism, limiting this method's sensitivity and specificity given theshelf life of the product (Brecher, Hogan et al. 1994). Even in light ofthese limitations many centers have moved to time of issue testsutilizing the measurement of pH and glucose with a handheld device, pHpaper, or in combination on a multi-reagent dipstick as surrogatemarkers for bacterial contamination (Burstain, Brecher et al. 1997;Yazer and Triulzi 2005).

In one method of non-invasive bacterial detection, changes in pH or theproduction of CO₂ was detected in clinical specimens by culturing thespecimens with a sterile liquid growth medium in a transparent sealedcontainer (Calandra et al., U.S. Pat. No. 5,094,955). The maindisadvantage of this method is the requirement for sampling of aclinical specimen and introduction of the sample into a separate culturevessel at one point in time. The sampling is disadvantageous because thecontaminating organisms may not be included in the sample volume,resulting in a false negative test. A similar method employed a sensorto detect microbial organisms growing in a liquid environment with themicrobial colonies immediately available for further testing by virtueof the design of the culture vessel (Jeffrey et al., U.S. Pat. No.5,976,827). This general method by which culture-based bacterialdetection systems function is currently used for detection ofmicroorganisms in PCs (Brecher, Means et al. 2001; McDonald, Pearce etal. 2005). A device for measuring the pH of PCs non-invasively by afluorescence-based interrogation of the bag contents has been describedin the WO 2006/023725 (PCT/US2005/029559), expressly incorporated hereinby reference in its entirety.

Additional important reasons to measure pH in the quality control of PCsinclude its correlation with in vivo viability following transfusioninto patients (Moroff, Friedman et al. 1982; Solberg, Holme et al. 1986;Holme 1998; Rinder, Snyder et al. 2003). pH Values below 6.2 in PCs havebeen correlated with poor in vivo recovery in transfusion studies(Murphy and Gardner 1971; Slichter and Harker 1976; Murphy 1985), whileloss of recovery in vivo at pH values above 7.2 has been shown (Murphyand Gardner 1975). The platelet yield in PCs is also an importantquality control parameter because it establishes the therapeutic dosageof the product and may influence the levels of metabolic activitymeasured within the storage bags. There have been several studiesshowing maintenance of pH values indicative of good platelet function upto 7 or 8 days in PCs stored in mixtures of additive solutions andplasma (Klinger 1996), provided that the platelet content was <4×10¹¹(de Wildt-Eggen, Schrijver et al. 1998). These latter studies showedthat a more rapid decline in pH in a PC may correlate with higherplatelet concentrations. A similar correlation of a more rapid declinein pH over the five day storage period was inferred in apheresis derivedPCs obtained from machines that consistently produce product with higherplatelet counts (Tudisco, Jett et al. 2005).

The storage of platelets in BTHC (n-butyryl, tri-n-hexyl citrate) PVCcontainers presents a number of advantages with respect to platelethealth. These containers have several desirable characteristics for thismedical application including low toxicity and permeability to water,O₂, and CO₂ in the desired ranges. The BTHC plasticized PVC materialalso has high permeability for CO₂, excess of which in some cases leadsto difficulties storing PCs which have high platelet counts. Dependingon storage conditions the pH of PCs can change rapidly due tooff-gassing of dissolved CO₂. The Council of Europe guidelines forplatelet storage conditions require the pH to be in the range of 6.4-7.4at 37° C. (6.6-7.6 at 22° C.).

Optical sensors (optrodes) for measuring pH are well known. Certainaromatic organic compounds (like phenolphthalein) change color with pHand can be immobilized on solid supports to form “pH paper.” Thesevisual indicators are easy to use, but do not provide a quantitativereading. The color changes can be difficult to distinguish accurately,and can be masked by colored analyte. Fluorescent indicators have alsobeen used as optical sensors. pH Sensitive fluorescent dyes can beimmobilized on solid supports and generally are more sensitive incomparison to the simple colorimetric (absorbance or reflectance based)indicators. The improved sensitivity of fluorescent indicators allowsthe solid support to be miniaturized, and this has been used toadvantage in development of fiber optic sensor devices for measuring pH,CO₂, and O₂ parameters in blood.

A specific need in the medical industry exists for accurate pHmeasurement of blood and blood products. The pH of blood or other bodilyfluids (pleural effusions) can be associated with certain physiologicresponses associated with pathology. Blood gas analyzers are commoncritical care instruments. Depending on storage conditions, the pH ofseparated blood components (plasma, platelets) can change rapidly due tooff-gassing of dissolved CO₂ from the enriched venous blood that iscollected from a donor. Platelets in particular are metabolicallyactive, and generate lactic acid during storage at 20° C. to 22° C.European quality guidelines for platelets prepared by the “buffycoatmethod” require pH of stored platelets to be pH 6.8-7.4 at 37° C.(7.0-7.6 at 22° C.).

Seminaphthofluorescein (SNAFL) compounds and the relatedseminaphthorhodafluor (SNARF) compounds are commercially availableratiometric fluors (Molecular Probes, Inc., Eugene, Oreg.; see, forexample, U.S. Pat. No. 4,945,171) and their synthesis and spectralproperties have been described. These compounds have advantagesincluding long wavelength absorbance that can be efficiently excitedwith LED light sources. Relevant acid/base equilibria and associatedspectral properties are shown below.

Deprotonation of the naphthol structure of SNAFL dyes gives anaphtholate molecule with longer wavelength fluorescence emission. ThepKa is the pH value where the two molecular species form in equalamounts. SNAFL compounds with reactive linker groups that allow theirconjugation to other molecules of interest are also commerciallyavailable.

Various methods have been used to immobilize “ratiometric” dyes to solidsupports for use in fiber optic pH detectors. Carboxynaphthofluorescein(CNF) has been conjugated to aminoethyl-cellulose and this material wasglued to polyester (Mylar) films to make sensing membranes for optrodes.The pKa of this material was determined to be 7.41, slightly lower thanthe free CNF (pKa 7.62). The use of tetraethoxysilane to trap CNF in asol-gel glass that was formed on glass cover slips has also beenreported. The pKa of this material was determined to be 7.46. A 9-chlorosubstituted SNAFL analog (SNAFL-2) has been reacted with polyvinylamineand the residual amino groups crosslinked with a photocrosslinker toform a gel-like coating on acrylic fibers. The pKa of this fiber-opticsensor was determined to be 7.14, significantly lower than the publishedpKa of the free SNAFL compound (pKa ˜7.7). This shows that molecularenvironment and linker structure surrounding the immobilized dye canalter the performance of a pH detector.

Despite the advances made in the detection of pH noted above, thereexists a need for improved methods and devices for monitoring thechemical environment in a sealed sterile container, such as a plateletstorage device, continually or at discrete time intervals, in order tobetter understand the types and levels of metabolic activities withinthe storage device, as well as their origin. The present invention seeksto fulfill this need and provides further related advantages.

SUMMARY OF THE INVENTION

The present invention provides a system and method for detecting andmeasuring chemical perturbations in a sample.

In one aspect, the invention provides a fluorescence method formonitoring a parameter in a sample. In one embodiment, the methodincludes:

(a) irradiating a fluorescent species having an emission that isdependent on a parameter with excitation light emanating from a probephysically isolated from the fluorescent species to provide afluorescent emission, wherein the fluorescent species is in liquidcommunication with a sample contained in a vessel;

(b) measuring the emission to determine a first parameter reading of thesample; and

(c) repeating step (a) after a pre-determined time and measuring theemission to determine a second parameter reading of the sample.

In one embodiment, step (c) is repeated to provide multiple parameterreadings over time to monitor the parameter of the sample. In oneembodiment, the parameter is pH. In another embodiment, the parameter isCO₂.

In one embodiment, the method further includes writing parameter data tothe vessel containing the sample, the vessel having a means forreceiving the parameter data that further allows the data to be readfrom the vessel at a later time.

In another aspect of the invention, a system for monitoring a parameteris provided. In one embodiment, the system includes:

(a) a light source for exciting a fluorescent species having an emissionthat is dependent on the parameter, wherein the fluorescent species isin liquid communication with a sample contained in a vessel;

(b) an emission detector for measuring emission from the fluorescentspecies and creating emission data;

(c) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(d) an emission lightguide for transmitting emission from thefluorescent species to the emission detector, wherein the lightguidecomprises a first terminus proximate to the detector and a secondterminus distal to the detector;

(e) a probe housing the distal termini of the excitation lightguide andthe emission light guide;

(f) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light, and wherein the window physically isolates the probefrom the fluorescent species;

(g) a memory device for storing emission data; and

(h) a processor device for converting the emission data to profile ofthe parameter over time.

In one embodiment, the system further includes a means for writingparameter data to the vessel containing the sample, the vessel having ameans for receiving the parameter data that further allows the data tobe read from the vessel at a later time.

In another aspect, the present invention provides a fluorescence methodfor monitoring the pH of a sample. In one embodiment, the methodincludes:

(a) irradiating a fluorescent species in liquid communication with asample contained in a vessel with excitation light emanating from aprobe physically isolated from the fluorescent species, wherein theexcitation light has a wavelength sufficient to effect fluorescentemission from the fluorescent species, wherein the fluorescent speciesexhibits a first emission intensity at a first emission wavelength and asecond emission intensity at a second emission wavelength, the ratio ofthe first and second emission intensities being dependent on pH;

(b) measuring the first and second emission intensities to determine thepH of the sample; and

(c) repeating step (a) after a pre-determined time and measuring thefirst and second emission intensities to determine the second pH of thesample.

In one embodiment, step (c) is repeated to provide multiple pHdeterminations to monitor the pH of the sample over time.

In one embodiment, the method further includes writing pH data to thevessel containing the sample. The vessel having a means for receivingthe pH data that further allows the data to be read from the vessel at alater time.

In another aspect of the invention, a system for monitoring pH isprovided. In one embodiment, the system includes:

(a) a light source for exciting a fluorescent species, wherein thefluorescent species has a first emission intensity at a first emissionwavelength and a second emission intensity at a second emissionwavelength;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(g) a probe housing the distal termini of the excitation lightguide,first emission lightguide, and second emission light guide; and

(h) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light, and wherein the window physically isolates the probefrom the fluorescent species.

In one embodiment, the system further includes a means for writing pHdata to the vessel containing the sample, the vessel having a means forreceiving the pH data that further allows the data to be read from thevessel at a later time.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 illustrates a representative large platelet storage bagincorporating a pH reading insert useful in the method and system of theinvention;

FIG. 2 illustrates a representative small platelet storage bagincorporating a pH reading insert useful in the method and system of theinvention;

FIGS. 3A-3D are graphs comparing pH profiles from different pHmeasurement systems for PC samples stored in representative plateletstorage bags;

FIG. 4A is a graph comparing Kunicki Scores for platelets stored inrepresentative and control platelet storage bags over a period of time;

FIG. 4B is a graph comparing Swirling Scores for platelets stored inrepresentative and control platelet storage bags over a period of time;

FIG. 5A is a graph comparing pH profiles of platelets stored inrepresentative and control platelet storage bags;

FIG. 5B is a graph comparing glucose and lactate concentration profilesof platelets stored in representative and control platelet storage bags;

FIG. 6A is a graph comparing ATP:ADP ratio profiles for platelets storedin representative and control platelet storage bags;

FIG. 6B is a graph comparing mitochondrial potential profiles forplatelets stored in representative and control platelet storage bags;

FIG. 7 is a graph comparing the level of platelet activation measured byCD62P and Annexin V percentages for platelets stored in representativeand control platelet storage bags;

FIG. 8 is a graph comparing pH profiles for under-filled andnormal-filled representative platelet storage bags;

FIGS. 9A-9D are graphs illustrating pH profiles for platelet samplescontaminated with different types of bacteria;

FIGS. 10A-10D are graphs illustrating the pH, glucose, pO₂, and pCO₂profiles, respectively, of platelet samples contaminated with bacteria.

FIG. 11 illustrates a representative multi-probe platelet storage baguseful in the method and system of the invention;

FIG. 12 is a schematic illustration of an automated single reader;

FIG. 13 is a schematic illustration of a multi-fiber reader;

FIG. 14 is a schematic illustration of an incubator;

FIG. 15 is a graph relating fluorescent intensity ratio to pH of PCs;

FIG. 16 is a graph relating fluorescence intensity ratios to blood gaspH of PCs;

FIG. 17 is a graph comparing pH profiles of various types of plateletsamples.

FIG. 18 is a schematic illustration of a representative system of theinvention for measuring pH;

FIG. 19 is a schematic illustration of an optical platform useful in thesystem of the invention for measuring pH;

FIG. 20 is a schematic illustration of a representative housing forexcitation and emission light guides useful in the system of theinvention;

FIG. 21 illustrates the relationship between the excitation/emissionoptical fiber housing and the sealed vessel port;

FIG. 22 is a representative port assembly useful in the manufacture of asealed vessel;

FIGS. 23A-23E illustrate the structures of representativeseminaphthofluorescein compounds useful in the method and system of theinvention;

FIG. 24 illustrates the emission spectra as a function of pH of arepresentative fluorescent species (SNAFL-1) useful in the method andsystem of the invention;

FIG. 25 illustrates the emission spectra as a function of pH of arepresentative fluorescent species (EBIO-3) useful in the method andsystem of the invention;

FIG. 26 is a schematic illustration of the preparation of arepresentative fluorophore-protein (EBIO-3/HSA) conjugate useful in themethod and system of the invention;

FIG. 27 illustrates the emission spectra as a function of pH of arepresentative fluorophore-protein conjugate (SNAFL-1/HSA) useful in themethod and system of the invention;

FIG. 28 illustrates the emission spectra as a function of pH of arepresentative fluorophore-protein conjugate (EBIO-3/HSA) useful in themethod and system of the invention;

FIG. 29 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (SNAFL-1/HSA) as afunction of pH (Oxyphen);

FIG. 30 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (SNAFL-1/HSA) as afunction of pH (nitrocellulose);

FIG. 31 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (EBIO-3/HSA) as afunction of pH (nitrocellulose);

FIG. 32 illustrates the data used in the method of the invention formeasuring pH;

FIG. 33 illustrates the results of the method of the invention forplatelet rich plasma;

FIG. 34 illustrates the correlation of pH results for platelet richplasma obtained by the method and system of the invention;

FIG. 35 illustrates stability of a representative substrate-immobilizedfluorophore conjugate of the invention;

FIG. 36 illustrates a representative device of the invention formeasuring carbon dioxide in a sealed vessel;

FIG. 37 illustrates the effect of probe position on fluorescentintensity in measuring pH in accordance with the invention; and

FIG. 38 illustrates the effect of membrane pore size on fluorescentintensity in measuring pH in accordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a system and method for detecting andmeasuring chemical perturbations in a sample. Perturbations in a sampleare detected and measured by monitoring the sample for changes in aparameter measurable by the system and method. Parameters measurable bythe system and method include pH and carbon dioxide (CO₂) level. Thesystem and method of the invention are useful for measuringperturbations in a blood sample or a blood product sample contained in asealed vessel.

In one aspect, the invention provides a fluorescence method formonitoring a parameter in a sample. In one embodiment, the methodincludes the steps of:

(a) irradiating a fluorescent species having an emission that isdependent on a parameter with excitation light emanating from a probephysically isolated from the fluorescent species to provide afluorescent emission, wherein the fluorescent species is in liquidcommunication with a sample contained in a vessel;

(b) measuring the emission to determine a first parameter reading of thesample; and

(c) repeating step (a) after a pre-determined time and measuring theemission intensity to determine a second parameter reading of thesample.

In one embodiment, step (c) is repeated to provide multiple parameterreadings over time to monitor the parameter of the sample. In oneembodiment, the parameter is pH. In another embodiment, the parameter isCO₂.

In one embodiment, the method further includes writing parameter data tothe vessel containing the sample, the vessel having a means forreceiving the parameter data that further allows the data to be readfrom the vessel at a later time.

In another aspect of the invention, a system for monitoring a parameteris provided. In one embodiment, the system includes:

(a) a light source for exciting a fluorescent species having an emissionthat is dependent on the parameter, wherein the fluorescent species isin liquid communication with a sample contained in a vessel;

(b) an emission detector for measuring emission from the fluorescentspecies and creating emission data;

(c) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(d) an emission lightguide for transmitting emission from thefluorescent species to the emission detector, wherein the lightguidecomprises a first terminus proximate to the detector and a secondterminus distal to the detector;

(e) a probe housing the distal termini of the excitation lightguide andthe emission light guide;

(f) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light, and wherein the window physically isolates the probefrom the fluorescent species;

(g) a memory device for storing emission data; and

(h) a processor device for converting the emission data to profile ofthe parameter over time.

In one embodiment, the system further includes a means for writingparameter data to the vessel containing the sample, the vessel having ameans for receiving the parameter data that further allows the data tobe read from the vessel at a later time.

In another aspect, the present invention provides a fluorescence methodfor monitoring the pH of a sample. In one embodiment, the methodincludes the steps of:

(a) irradiating a fluorescent species in liquid communication with asample contained in a vessel with excitation light emanating from aprobe physically isolated from the fluorescent species, wherein theexcitation light has a wavelength sufficient to effect fluorescentemission from the fluorescent species, wherein the fluorescent speciesexhibits a first emission intensity at a first emission wavelength and asecond emission intensity at a second emission wavelength, the ratio ofthe first and second emission intensities being dependent on pH;

(b) measuring the first and second emission intensities to determine thepH of the sample; and

(c) repeating step (a) after a pre-determined time and measuring thefirst and second emission intensities to determine the second pH of thesample.

In one embodiment, step (c) is repeated to provide multiple pHdeterminations to monitor the pH of the sample over time.

In one embodiment, the method further includes writing pH data to thevessel containing the sample, the vessel having a means for receivingthe parameter data that further allows the data to be read from thevessel at a later time.

In the above methods, the parameter profile or pH profile is made bymultiple parameter or pH measurements. The multiple measurements aremade over time and establish the profile. When the interval of timebetween measurements is sufficiently small, the profile revealsperturbations, if any, in the sample over the time period of theprofile. In the methods of the invention, the interval of time betweenmeasurements is pre-determined. The pre-determined times are set toreveal perturbations in the sample.

In one embodiment, the pre-determined time is a variable time periodfrom 1 minute to 1 day. In one embodiment, the pre-determined time isfrom between 1 hour to 12 hours.

In the methods, the multiple parameter or pH determinations over timeprovide a parameter or pH profile of a sample. The parameter or pHprofile of the sample can be used to determine a quality of the sample.

In the above methods, the probe is physically isolated from thefluorescent species. As used herein, the term “physically isolated”refers to the physical isolation of the probe from the sample beinginterrogated. The probe providing excitation light and receivingemission light does contact the sample being interrogated. In themethods, the sample is in contact (i.e., liquid communication) with thefluorescent species (e.g., substrate-immobilized fluorescent species).The probe is isolated from and does not come not physical contact withthe sample. The isolation of the probe from the sample is illustrated inFIG. 21. The probe is isolated from the fluorescent species by a windowtransparent to the excitation light and the fluorescent emission.

In another aspect of the invention, a system for monitoring pH isprovided. In one embodiment, the system includes:

(a) a light source for exciting a fluorescent species, wherein thefluorescent species has a first emission intensity at a first emissionwavelength and a second emission intensity at a second emissionwavelength;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(g) a probe housing the distal termini of the excitation lightguide,first emission lightguide, and second emission light guide; and

(h) a housing for receiving the probe, wherein the housing is adaptedfor receiving the probe at a first end and terminating with a window atthe second end, the window being transparent to the excitation and theemission light, and wherein the window physically isolates the probefrom the fluorescent species.

In one embodiment, the system further includes a means for writing pHdata to the vessel containing the sample, the vessel having a means forreceiving the pH data that further allows the data to be read from thevessel at a later time.

In one embodiment, the first and second detectors are the same. In oneembodiment, the first and second detectors are different. In oneembodiment, the first and second detectors are photodiodes.

In one embodiment, the first emission lightguide and the second emissionlightguide are the same. In one embodiment, the first emissionlightguide and the second emission lightguide are different. In oneembodiment, the excitation lightguide, the first emission lightguide,and the second emission lightguide are optical fibers.

There are five basic optical chemical sensing techniques: measuringabsorbance, fluorescence intensity, ratiometric fluorescence,fluorescence lifetime, and fluorescence polarization. Sample parameters,such as pH, can be measured using any of the five techniques. Thesystems and methods of the invention utilize fluorescence sensing andcan be used to monitor sample parameters that produce changes influorescence intensity, ratiometric fluorescence, fluorescence lifetime,and fluorescence polarization characteristics of a fluorescent speciesin liquid contact with the sample to be monitored.

In one embodiment, the method is a fluorescent wavelength ratiometricmethod. As used herein, the term “fluorescent wavelength-ratiometric”refers to a method by which the first and second fluorescent emissionintensities of a fluorescent species are measured at first and secondemission wavelengths, respectively, and are ratioed to provide pHinformation. In this embodiment, the fluorescent species has emissionintensities that vary as a function of pH.

In one embodiment of the method, the fluorescent species is aratiometric fluorescent species. In one embodiment, the fluorescentspecies is selected from a naphthofluorescein compound and aseminaphthorhodamine compound. In one embodiment, the naphthofluoresceincompound is selected from a seminaphthofluorescein compound and acarboxynaphthofluorescein compound. In one embodiment, theseminaphthofluorescein compound is selected from 5′(and6′)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-one(also referred to herein as “SNAFL-1”, see FIG. 23A) and2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid (also referred to herein as “EBIO-3”, see FIG. 23E).

In one embodiment, the fluorescent species is immobilized on a substrateand comprises a conjugate of a fluorescent species and a macromolecule.In one embodiment, the macromolecule is an albumin. In one embodiment,the macromolecule is a serum albumin. In one embodiment, themacromolecule is a human serum albumin. In one embodiment, themacromolecule is a recombinant human serum albumin. In one embodiment,the fluorescent species immobilized on a substrate comprises anaphthofluorescein/serum albumin conjugate. In one embodiment, thefluorescent species immobilized on a substrate comprises aseminaphthofluorescein/human serum albumin conjugate.

As noted above, the method is suitable for measuring the pH of blood orblood products contained in a sealed vessel. In one embodiment, thefluorescent species (e.g., substrate-immobilized fluorescent species) isintroduced into a sealed vessel during the vessel's manufacture andbefore the sample is introduced to the vessel. In one embodiment, thefluorescent species is introduced into a sealed vessel by a means thatpreserves the vessel's seal.

The present invention provides a method and system for monitoring aparameter of a sample. The method and system are useful in monitoringthe pH or CO₂ of blood or blood products. The method and system aresuited to monitor the pH of a sample contained in a sealed vessel.

The presence of microorganisms growing in PCs causes dynamic changes invarious parameters in the mixture, such as pH, CO₂, O₂, and glucoseconcentrations. These parameters can be analyzed as surrogate markersfor the presence of microbiological contamination. A non-invasive“in-the-bag” sensor, used to measure chemical parameters such as pH, CO₂levels, O₂ levels, or glucose concentrations provides greatersensitivity for detecting perturbations in the bag environment caused bythe growth of microorganisms due to the elimination of samplingartifacts and the ability to carry out multiple measurements over timewithin a closed system. The systems and methods of the invention candetect subtle changes in pH or CO₂ within the closed system or the bagenvironment, and can be monitored continuously or periodically over timewithout direct sampling.

In one embodiment, the invention provides a method of monitoring pH of asealed sterile platelet storage device over time and analyzing thechanges to predict the quality of the platelet rich plasma and/orplatelet rich plasma additive solution formulations. The non-invasivemethods described herein enable this “real-time” analysis of plateletquality. Previously, samples had to be physically withdrawn from theplatelet storage bag and analyzed on a blood gas analyzer or a separateinstrument. After puncturing the bag, the PC must be used within 4 hoursdue to risk of contamination. Preserving the unit required laborioussampling of bag contents via a long plastic tube attached to the bag(pigtail) using a sterile sealer device to remove portions thereof.

The data were presented herein was obtained using a small plateletstorage bag with a 14 mL storage volume having a built-in pH readinginsert. A larger storage bag for a transfusible unit is designed to hold300 mL of pooled platelet concentrate. This larger bag could be used forplatelets recovered from a single donor by apheresis or for storage of apre-storage pooled platelet concentrate unit.

Large and small platelet storage bags with built-in inserts are shown inFIGS. 1 and 2, respectively. Referring to FIG. 1, large storage bag 500includes a plurality of vessel ports 510 and port assembly 232. Portassembly 232 is further described in FIG. 22. Storage bag 500 alsoincludes label 1300, into which a memory device can be integrated (notshown). By integrating a memory device into label 1300, parameterreadings can be written to and stored on storage bag 500 that containsthe sample.

Referring to FIG. 2, small storage bag 500 includes a plurality ofvessel ports 510 and port assembly 232. Port assembly 232 is furtherdescribed in FIG. 22. Storage bag 500 also includes label 1300, intowhich a memory device can be integrated (not shown). By integrating amemory device into label 1300, parameter readings can be written to andstored on storage bag 500 that contains the sample.

The pH reading accuracy of the non-invasive platelet bag inserts andmeasuring system described herein have been tested. FIGS. 3A-3D aregraphs showing pH profiles of four individual PC samples. The PCs werestored in representative large sterile storage bags containing 300 mL ofpooled platelet concentrates per bag. These platelets were leukoreducedprior to storage in the bags, and the four bags tested all showed goodplatelet health parameters over the 11 days of storage. The bags werespiked in an aseptic manner with sample site adaptors (Codan) via thetwist off ports incorporated on the bags to allow samples to bewithdrawn for analysis on a blood gas analyzer.

Referring to FIGS. 3A-3D, two pH profiles are shown for each sample. ThepH profiles for each sample were generated from two pH measurementsystems, namely, a non-invasive fluorescent pH reading and an invasiveblood gas pH reading. In each figure, the pH profile generated fromfluorescent pH readings is designated “BCSI pH” and shown as a solidline. The pH profile generated from blood gas pH readings is designated“Blood Gas pH” and shown as a dashed line. The data show similar pHprofiles for these “normal” platelet concentrates over 11 days ofstorage. This demonstrates that the bags with inserts can be ethyleneoxide sterilized without compromising pH reading performance. This alsodemonstrates that the accuracy of the two pH measurement systems(non-invasive fluorescent reading vs. invasive blood gas reading) wasconsistent. The mean difference from average pH determined by bothmethods was 0.045 (n=32, SD 0.058). This is the first demonstration of asystem for measuring the metabolic activity in the contents of aplatelet storage bag in a non-invasive manner over time.

To determine the suitability of the representative manufactured bags forstorage of platelet concentrates, the four sterile representative bagsdescribed above were compared with similar sized control sterileplatelet storage bags from Fresenius (The Netherlands). Two 300 mLpooled platelet rich plasma units were leukoreduced, combined anddistributed to the paired storage bags. The bags were spiked in anaseptic manner with sample site adaptors (Codan) via the twist off portsincorporated on the bags to allow samples to be withdrawn for analysison a blood gas analyzer. The results, shown in FIGS. 4-7, demonstratevirtually identical platelet storage parameters for the pairedrepresentative (“BCSI”) and control (“Fresenius”) bags.

Referring to FIGS. 4A and 4B, platelet shape parameters for PCs storedin representative and control platelet storage bags were evaluated. FIG.4A is a graph comparing Kunicki Scores of PCs stored in representativeand control platelet storage bags for 9 days. The Kunicki Scores in FIG.4A range in value from 0 to 400. A Kunicki Score of 0 indicatesballoon-shaped, or dead platelets; 100 indicates dendrites, or anactivated platelets; 200 indicates spheres, or spherical platelets; and400 indicates discoid platelets. The results show virtually identicalstorage parameters for the paired representative and control bags.

FIG. 4B is a graph comparing Swirling Scores for platelets stored inrepresentative and control platelet storage bags for 9 days. TheSwirling Scores in FIG. 4B range in value from 0 to 3. A Swirling Scoreof 0 indicates dead platelets; a score of 2 is the standard value forplatelets at 6 days of storage; and a score of 3 indicates very goodplatelets. The results show virtually identical storage parameters forthe paired representative and control bags.

Referring to FIGS. 5A and 5B, the pH, glucose, and lactate profiles forPCs stored in representative and control platelet storage bags weredetermined. FIG. 5A is a graph comparing pH profiles of platelets storedin representative and control platelet storage bags for 9 days. Theresults show virtually identical storage parameters for the pairedrepresentative and control bags.

FIG. 5B is a graph comparing glucose and lactate concentration profilesof platelets stored in representative and control platelet storage bagsfor 9 days. The glucose concentrations are depicted with diamond-shapedpoints and range from approximately 20 mM at storage day 1 toapproximately 10 mM at day 9. The lactate concentrations are depictedwith triangle-shaped points and range from approximately 6 mM at storageday 1 to approximately 17 mM at day 9. The results show virtuallyidentical storage parameters for the paired representative and controlbags.

Referring to FIGS. 6A and 6B, the metabolic function of platelets storedin representative and control bags was determined. FIG. 6A is a graphcomparing ATP:ADP ratio profiles for platelets stored in representativeand control platelet storage bags for 9 days. The ATP:ADP ratio ismeasured on a scale of 1 to 10. An ATP:ADP ratio of 1 indicates aresting sample, and a ratio of 10 indicates a metabolically activesample. The results show a typical ATP:ADP response for the pairedrepresentative and control bags.

FIG. 6B is a graph comparing mitochondrial potential profiles forplatelets stored in representative and control platelet storage bags for9 days. The mitochondrial potential is measured on a scale of 0 to 4. Amitochondrial potential reading of 0 indicates damaged platelets, and amitochondrial potential reading of 3 indicates good platelets. Theresults show good mitochondrial potential throughout the test period forthe paired representative and control bags.

Referring to FIG. 7, platelet activation levels were measured for PCs inrepresentative and control platelet storage bags for 9 days. FIG. 7 is agraph comparing the level of platelet activation measured by CD62P andAnnexin V percentages. The CD62P percentages are depicted with thediamond-shaped points and range from approximately 5% at storage day 2to approximately 17% at day 9. The Annexin V percentages are depictedwith triangle-shaped points and range from approximately 5% at storageday 4 to approximately 16% at day 9. The results are consistent withmoderate platelet activation in paired representative and control bags.

To further demonstrate the utility of the invention, pH over time forplatelets stored in representative small platelet storage bags wasexamined. Referring to FIG. 8, pH profiles were generated using themethod and system of the invention for normal and under-filled smallplatelet storage bags. The normal bags contained 14 mL of apheresisplatelets. The under-filled bags contained 7 mL of apheresis plateletsfrom the same unit as the normal bags. The best storage conditions used14 mL of PC per bag, and reproducible curves were obtained for these“normal” storage conditions. As shown in FIG. 8, if the bags areunder-filled (7 mL of PC per bag), then the pH profile changes such thatthe pH drops more rapidly. This is the first demonstration that thenon-invasive system for measuring the metabolic activity in the contentsof a platelet storage bag over time can be used to find “abnormal”units. Other storage parameters such as abnormal leukocyte count orabnormal platelet concentration or novel parameters that influenceplatelet storage may also be detected using the non-invasive method ofthe invention. It is recognized that platelets recovered from somedonors rapidly lose function and it is likely that these units can bediscovered using a non-invasive system.

Bacterial contamination of platelets is a common problem, and the mostlife threatening of all blood transfusion events. Results ofnon-invasive pH measurements of small platelet bags inoculated with fourcommon bacterial contaminants are shown in FIGS. 9A-9D. The bags wereinoculated at 26 hours and detected at the inflection time point shownin each graph. All bags were incubated at 22° C. FIG. 9A represents a PCsample inoculated with 10 CFU/mL (Colony Forming Units/mL) of E. coli.The sample at 72 hours contained 3.7E4 CFU/mL bacteria. FIG. 9Brepresents a PC sample inoculated with 10 CFU/mL of K. oxytoca. Thesample at 48 hours contained 3.3E5 CFU/mL of bacteria. FIG. 9Crepresents a PC sample inoculated with 90 CFU/mL S. marcescens. Thesample at 48 hours contained 3.6E3 CFU/mL of bacteria. FIG. 9Drepresents a PC sample inoculated with 240 CFU/mL S. aureus. The sampleat 60 hours contained 6.2E7 CFU/mL of bacteria.

Referring to FIGS. 9A-9D, pH was measured using a Bayer blood gasanalyzer and a representative system and method of the presentinvention. All organisms tested in this cohort show a dramatic decreasein pH with ongoing bacterial growth. Concomitant invasive sampling withthe blood gas analyzer confirmed the accuracy of the non-invasive pHmeasurement system. The “detection point” shown in FIGS. 9A-9D can becorrelated with bacterial count upon examination of the rate of changein pH per unit of time over the course of the bag tracking pHmeasurements. The “detection point” can also be correlated with otherchemical parameters or numerical ratios thereof as measured usingvarious methods in these experiments. More frequent sampling wouldlikely detect the bacteria at lower CFU.

The value of measuring other analytes associated with metabolizingplatelets in storage bags is shown in FIGS. 10A-10D. Small storage bagswere inoculated with 10 CFU/mL of K. pneumoniae. Partial pressures ofoxygen and carbon dioxide were measured on a blood gas analyzer atvarious timepoints. Glucose concentrations were measured using acommercial dipstick method at the same timepoints. The pH measurementsin FIG. 10A show paired blood gas and pH readings for a singleinoculated bag. The O₂, CO₂, and glucose graphs in FIGS. 10C, 10D, and10B, respectively, compare inoculated and “normal” plateletconcentrates. Bacterial contamination of the platelet concentrates canalso be correlated with these other chemical parameters, or numericalratios thereof, as measured using various methods in these experimentsas shown in FIGS. 10A-10D.

FIG. 11 illustrates a representative multi-probe platelet storage bagwith two inserts, one for measuring pH and the other for measuring CO₂.Referring to FIG. 11, storage bag 500 includes a plurality of vesselports 510, port assembly 232, and port assembly 232. Storage bag 500includes label 1300, into which a memory device can be integrated (notshown). By integrating a memory device into label 1300, parameterreadings can be written to and stored on storage bag 500 that containsthe sample.

Thus, in one embodiment, the invention provides a method of monitoring(e.g., measuring and recording) at least two chemical parameters in asealed sterile platelet storage device. In the method, the parametersmonitored indicate platelet healthiness (e.g., low pH) or microbialcontamination (e.g., CO₂ spike).

Storage of the platelet units at 22° C. in a shaking incubator isstandard in all blood banks worldwide. Although fluorescence readers canbe used to rapidly measure pH, it still requires individual handling ofevery bag at each time point. To alleviate this problem, in oneembodiment, the invention provides an incubator that facilitatesautomated reading.

In one embodiment, a single reader incubator with a flexible probe isprovided. The probe is mechanically moved to different positions on theback of the incubator using an x,y,z robot. Referring to FIG. 12, anoptical platform 180 is located proximate to a shelving unit that isconfigured to receive platelet storage bags 500. The optical platform180 is more fully described below (see FIG. 19). Platelet storage bags500 are placed on shelves 5100 by a user. A plurality of apertures 5200are disposed towards the distal end of each shelf. Clamps 5300 arelocated at each aperture 5200. Each bag 500 may be secured through anaperture 5200 using clamp 5300. A probe 185 is connected by opticalfibers (light guides) to optical platform 180. Probe 185 is enabled tomaneuver within an x,y,z grid. The z dimension involves reversibleinsertion of the probe 185 into the clamped insert for each bag 500 at aspecified x,y position. Any number of bags may be placed in theincubator provided there are sufficient apertures 5200 to receive them.

In another embodiment, a single reader incubator is configured withmultiple fiber bundles. Referring to FIG. 13, multiple bags 500 withsensors are each connected to separate probes 185 by a user and leftattached to the probes. Separate probes 185 are connected to opticalplatform 180 by optical fibers (light guides). Optical platform 180 ismore fully described below (see FIG. 19). The fiber optics in the probes185 convey excitation light to the sensor and fluorescent emissions backto the detector within the optical platform 180. The excitation lightcan be split from one light source and piped to multiple probes 185 andbag sensors by physically blocking the excitation fiber optics so thefiber optic for only one probe is excited.

For example, if four probes (1, 2, 3, and 4) are connected to a singleexcitation source a moving shutter blocks the light collection ends ofprobes 1, 2 and 3 to only allow light to travel to probe 4. Then theshutters are changed to block the ends of probes 2, 3, and 4 allowingexcitation only to probe 1. Each of the dual emissions from the sensorsare collected with light for each channel from all the probes focused ona single detector. For example, a system with four probes (1, 2, 3, and4) and emission readings A and B for each probe, would have twodetectors A and B. Detector A would have all the light from probe 1A,2A, 3A, and 4A focused on it and likewise with detector B. Thendepending on what probe was excited by means of the physical blocking,the emission readings can be tied to which probe was excited. Aplurality of bags could be studied with a single excitation and emissionsystem in this way also a plurality of these excitation and emissionsystems 700 could be incorporated into a single central processor 170,with output to user interface and display 130.

FIG. 14 is a schematic illustration for the incubators described inFIGS. 12 and 13. Referring to FIG. 14, the schematic illustration showshow the components of a representative system for carrying out themethod of the invention are adapted and connected to make an incubator.System components include display 130 for determining the status of thesystem and viewing pH determination results; memory device 165 forstoring test results and calibration data; signal processing electronics170 for commanding the optical platform components and processingsignals from the optical platform; and optical platform 180 including anexcitation source 280, emission detectors 380 and 480, light guides, andassociated lenses and filters. Optical platform 180 includes probe 185.Probe 185 is reversibly connectible to bag 500 with insert.

In one embodiment, an incubator machine may have one probe 185 thatconnects to multiple bags 500, as shown in FIG. 12. In anotherembodiment, an incubator machine may have multiple probes 185 and asingle optical platform 180, as shown in FIG. 13. In yet anotherembodiment, an incubator machine may have multiple probes 185 withmultiple excitation sources 280 and emission detectors 380 and 480. Thebasic structure for this embodiment is shown in FIG. 14.

To have practical utility, the parametric information (e.g., pH)obtained by the systems and methods of the invention need be stored andanalyzed. There are several options for storing and analyzing theobtained data. Multi-read data can be stored for real-time analysis on amemory device, such as a chip, that is integrated into the storage bag(i.e., vessel) itself. The memory device is read/write capable in orderto receive information and store it for later analysis. In this way,data are directly linked to the bag and follow the bag from one locationto the next. For example, a bag may be transferred from a donor centerto a research laboratory or hospital blood bank. Storing the data on amemory device that is integrated into the bag allows the data to be readdirectly from the storage bag prior to being analyzed.

In one embodiment, data of the pH and other analytes can be stored on amemory device attached to the platelet bag. A Texas Instruments 52000RFID transponder and corresponding low-frequency RFID tags were employedas a proof of principle platform. The available RFID tags were limitedin memory capacity, having at most 80 bits available for read/writeoperations. To overcome this memory limitation, pH values were encodedto have a range such that pH 6.2 and 7.8 corresponded to hexadecimalvalues zero (0) and fifteen (F), respectively. In binary representationeach hexadecimal value requires four bits.

The representative system of the invention (BCSI pH1000, Blood CellStorage, Inc., Seattle, Wash.) was coupled to the RFID system through apersonal computer. An RFID antenna was fitted inside the pH1000 casing.Software was written in Matlab to integrate the functions of the pH1000and S2000. When a transponder entered the antenna range, data wasautomatically read from the RFID tag and the user prompted to perform areading. Subsequent to additional measurements on the pH1000; new datawas appended to the RFID tag.

Bags can be identified and tracked by integrating an RFID chip in thebag itself as a means for writing data to the vessel containing thesample. Referring to FIGS. 1 and 2, the chip can be integrated in thelabel 130 of bag 500. Bags can also be identified and tracked byidentifying each bag with a unique bar code. The bar code on theplatelet storage bag can also be used for other identificationinformation, for example, to identify the bag's position in theincubator. When data are stored on an RFID chip, there is no need toidentify the bag position in the incubator.

Another data storage option is to store data on a central computer thatis linked to the system for monitoring pH. When data are collected, theyare transferred to a central computer where they are stored. The centralcomputer is equipped to generate a pH profile using data that it hasaccumulated. Yet another option is to store data directly on the reader.Data can be processed on the reader to generate a pH profile, or can betransferred to a central computer that generates the pH profile.

To advise regarding the quality of the sample in the vessel interrogatedby the systems and methods of the invention, pass/fail algorithms may beutilized that examine the rate of change in pH per unit of time over thecourse of the bag tracking in an automated fashion. A drop in pH greaterthan some threshold value as defined from examination of a plurality ofdata would constitute assignment and reporting of a FAIL value whichwill be associated with the particular bag being examined.

In one aspect of the invention, a method for monitoring pH of a sampleis provided. In the method, pH is determined by comparing fluorescentemission intensities from a single fluorescent species havingpH-dependent fluorescent emission. The fluorescent species havingpH-dependent fluorescent emission has a first emission intensity at afirst wavelength and a second emission intensity at a second wavelength,the first and second emission intensities being characteristic of pH inthe environment of the fluorescent species. The ratio of the first andsecond emission intensities provides pH measurement. Calibration of thefirst and second emission intensities provides an intensity-basedreference (ratio information) that is used to determine the pH of theenvironment of the fluorescent species.

In one embodiment, fluorescent signal analysis of the ratio of first andsecond emission wavelengths is used to calculate the resulting pH.Fluorescent signal analysis and analysis of the pH algorithm areperformed as follows. The instrument collects the fluorescentintensities at 568 nm (fr) and at 600 nm (fp), and then calculates theratio of the fp to fr signals. The pH has been found to be a function ofthe ratio and the fp value described as pH=f(ratio)+g(fp). The f(ratio)function is nonlinear while the g(fp) function is linear. The bestdescription of the f(ratio) is Constant1*ln(ratio)+Constant2. Theinstrument then uses a lookup table, such as the lookup table in FIG.15, to find the appropriate value for the f(ratio) function and thencalculates the g(fp) adjustment and calculates a pH. Because theintensities of the fr and fp channels are dependent on optical path andthe instrument hardware, the intensity values obtained from a singlepart are not identical for each instrument. However, the differencesbetween these intensities are linearly related and thus it is possibleto compare the results from one instrument to another as well as createthe f(ratio) and g(fp) relationships on a different instrument than theinstrument that the calibration of pH and fluorescent signal wasperformed on.

FIG. 15 is a graph of a mathematical function of the ratio of first andsecond emission intensities as related to pH. This type of graph is alsoknown as a lookup table, and is useful in the method and system of thepresent invention. For example, according to the lookup table in FIG.15, a ratio of 1.6 corresponds to pH 6.0; a ratio of 2.56 corresponds topH 7.0; and a ratio of 4.48 corresponds to pH 8.0.

The lookup table in FIG. 15 is generated from calibration data depictedin FIG. 16. FIG. 16 shows the data from calibration studies of fivelots. Individual lookup tables are possible for each lot or as FIG. 16demonstrates the lots when compared to one lookup table agree within+/−0.159 pH units. Calibration data is collected in the manner describedin Example 7. Apheresis platelets and plasma are separated into largeplatelet bags rigged to resemble the small bag (see FIG. 2). Four groupsare followed over time to give a suitable range of pH. The four groupsare: (1) normal—standard fill of 12-15 mLs to generate pH decline overthe shelf life of the platelets, (2) plasma—to generate high pH range,(3) underfills—fill of only 7 mLs, and (4) bacterially spiked—standardfill with the addition of 100 CFU/mL of Klebsiella oxytoca after the 24hour read to generate very low pH. Readings are initially performedafter 4 hours of equilibration, then twice daily through four days andonce daily for the remainder of 7-10 days.

Platelet pH trends were analyzed to determine acceptable andunacceptable rates of change for stored platelets. The rate of changecan be used to determine the quality of stored PC. Referring to FIG. 17,pH profiles were generated for 5 types of platelets: PC sample with alow platelet count, PC sample with a high platelet count, two PC sampleswith bacterial contamination, and PC sample that is a “poor storer.”After an initial rise in pH attributable to CO₂ off gassing, pH rates ofchange can be described as follows. PC samples with a high plateletcount had a pH drop of about 0.02 pH units per day. PC samples with alow platelet count had a pH drop of about 0.04 pH units per day. pHdrops in this range are characteristic of good quality PCs that areusable. In comparison, poor storing PCs had a pH drop of about 0.08 pHunits per day, and bacterially-contaminated PCs had a pH drop of about0.2 pH units per day. pH drops in this range are characteristic of poorquality PCs that are unusable.

To further understand the systems and methods of the invention, thefollowing detailed description is provided.

In one embodiment, the fluorescent species having pH-dependentfluorescent emission is immobilized on a substrate in contact with thesample such that the fluorescent species is in contact with the sample.The immobilized fluorescent species in contact with the sample islocated in the sample such that the fluorescent species can beinterrogated. The fluorescence measurement is made by irradiating thefluorescent species at a wavelength sufficient to elicit fluorescentemission, which is then measured. Because of the pH-dependent nature ofthe fluorescent species' emission profile (i.e., first and secondfluorescent emission intensities measured at first and second emissionwavelengths, respectively) the measurement of the fluorescent emissionprofile yields the pH of the fluorescent species' environment (i.e.,sample pH).

In one embodiment, the sample for which the pH is to be determined iscontained in a sealed vessel. As noted above, this method is suitablefor measuring pH of blood and blood products sealed in a conventionalblood storage vessel.

In another embodiment, the sample for which the pH is to be determinedis contained in an open vessel. As used herein, the term “open vessel”refers to a vessel that is not sealed. In this method, the probe iscleaned and/or sterilized and is used once and discarded. This method issuitable for measuring the pH of materials used in food, pharmaceutical,or biological research where the vessel containing the material is notsealed (i.e., open). Such a “lab-use” system includes a tip (seedescription below) placed onto the probe. The pH measurement is made byimmersing the tip into the sample and measuring pH. The tip is removedfrom the sample, removed from the probe, and discarded.

In the method for measuring the pH of a contained sample, thefluorescent species (e.g., substrate-immobilized fluorescent species) isintroduced into the vessel either before or after the sample is placedin the vessel. The sealed vessel prevents the contents of the vesselfrom contact from, for example, liquids, gases, or contaminants outsideof the vessel. The sealed vessel also prevents the contents of thevessel from escaping the vessel.

The vessel can be manufactured to include the substrate-immobilizedfluorescent species as a component of the vessel. In such an embodiment,the substrate-immobilized fluorescent species is incorporated into thevessel during manufacture to provide a vessel into which a sample can belater introduced and its pH measured. The manufacture of a vesselincorporating the substrate-immobilized fluorescent species is describedin Example 1.

Alternatively, the substrate-immobilized fluorescent species can beintroduced into the vessel after the sample has been introduced into thevessel. In such an embodiment, the substrate-immobilized fluorescentspecies is introduced into the vessel by a process in which the vesselis first punctured (or spiked) to introduce the substrate-immobilizedfluorescent species and then resealed to provide a sealed vesselincluding the sample now in contact with the substrate-immobilizedfluorescent species. The process for introducing thesubstrate-immobilized fluorescent species into a sealed vessel isdescribed in Example 2.

As noted above, the vessel including the substrate-immobilizedfluorescent species in contact with the sample is sealed before, during,and after interrogation. Interrogation of the fluorescent speciesrequires excitation of the species at a wavelength sufficient to effectfluorescent emission from the species and measurement of thatfluorescent emission. In the method of the invention, interrogation isaccomplished through a window in the sealed vessel. The fluorescentspecies is excited by irradiation through the window, and emission fromthe fluorescent species is collected from the fluorescent species thoughthe window. The window is a component of the sealed vessel and allowsfor interrogation of the fluorescent species in contact with the sample.The window is sufficiently transparent at the excitation and emissionwavelengths to permit interrogation by the method. Thesubstrate-immobilized fluorescent species is positioned in proximity tothe window sufficient for interrogation: proximity sufficient toeffectively excite the fluorescent species and to effectively collectemission from the fluorescent species. It will be appreciated that forepifluorescence applications, a single window is used. However, othermethods and devices of the invention can include other optical paths,such as straight-through or right angle optical paths, where more thanone window can be used.

The method of the invention includes irradiating the fluorescentspecies, which in one embodiment is contained along with a sample in asealed vessel, at a wavelength sufficient to effect emission from thefluorescent species and to measure that emission. Exciting light andfluorescent emission pass through the sealed vessel's window. In oneembodiment, the sealed vessel further includes a port for receiving ahousing that holds the excitation light guide and emission light guide.In one embodiment, the excitation light guide includes one or moreoptical fibers that transmit the excitation light from a light source tothe fluorescent species. In one embodiment, the emission light guideincludes one or more optical fibers that transmit the emission lightfrom the fluorescent species to a light detector. The port receiving thehousing is positioned in proximity to the window sufficient forinterrogation: proximity sufficient to effectively excite thefluorescent species and to effectively collect emission from thefluorescent species.

As with all optical fluorescent methods, the method of the inventionincludes a light source for exciting the fluorescent species and adetector for measuring the emission of the fluorescent species. Lightsources, wavelength selection filters, and detectors are selected basedon the absorbance and emission profiles of the fluorescent species usedin the method.

Suitable light sources provide excitation energy at a wavelength andintensity sufficient to effect fluorescent emission from the fluorescentspecies. The light source can provide relatively broad wavelength bandexcitation (e.g., ultraviolet or white light sources) or relativelynarrower wavelength band excitation (e.g., laser or light-emittingdiode). To enhance excitation efficiency and emission measurement,relatively broad wavelength band exciting light from the source can beselected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitablelight sources include tungsten lamps, halogen lamps, xenon lamps, arclamps, LEDs, hollow cathode lamps, and lasers.

Suitable detectors detect the intensity of fluorescent emission over theemission wavelength band of the fluorescent species. To enhance emissionmeasurement, fluorescent emission from the fluorescent species sourcecan be selected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitabledetectors include photomultiplier tubes and solid state detectors, suchas photodiodes, responsive to the wavelength emission band of thefluorescent species. Other suitable detectors are photovoltaic cells,PIN diodes, and avalanche photodiodes.

Through the use of filters, all of the excitation light that reflectsoff the target is filtered out before reaching the detector. This can beachieved by using filters in both the excitation and emission opticalpaths. In certain instances, reflected excitation light (which is manyorders of magnitude more intense than the emission light) that reachesthe detector can swamp the specific signal. Generally, 10E5 (10⁵) orgreater out-of-band rejection is appropriate in each of the filter sets.Reduction of excitation light can also be achieved by using an angledwindow so that reflected light is directed away from the emissiondetector. However, such an optical path is not as effective as filtersets.

Excitation light from the source can be directed to the fluorescentspecies through the use of a light guide, such as one or more opticalfibers. Similarly, emission from the fluorescent species can be directedto the detector through the use of a light guide, such as one or moreoptical fibers.

A representative system for carrying out the method of the invention isillustrated schematically in FIG. 18. Referring to FIG. 18, system 100includes controller 110 that controls and operates the systemcomponents. System components include keypad 120 for inputtinginformation including system commands; display 130 for determining thestatus of the system and viewing pH determination results; barcodereader 140 for inputting information to the system including theidentification of the sample, the pH of which is to be measured by thesystem; printer 150 for printing system status and pH determinationresults; battery (or wall plug and power adapter) 160 for powering thesystem; memory device 165 for storing test results and calibration data;signal processing electronics 170 for commanding the optical platformcomponents and processing signals from the optical platform; and opticalplatform 180 including an excitation source, emission detectors, lightguides, and associated lenses and filters. Optical platform includesprobe 185 housing one or more excitation light guides and two or moreemission light guides. FIG. 19A also illustrates sealed vessel 500including port 205 for receiving probe 185.

FIG. 19 is a schematic illustration of an optical platform useful in thesystem of the invention for measuring pH. Referring to FIG. 19, opticalplatform 180 includes excitation optics 280, first emission optics 380,and second emission optics 480. Excitation optics 280 include lightsource 282, collimating lens 284, filter 286, focusing lens 288, andexcitation light waveguide 290. First emission optics 380 includedetector 382, focusing lens 384, filter 386, collimating lens 388, andfirst emission light waveguide 390. Second emission optics 480 includesdetector 482, focusing lens 484, filter 486, collimating lens 488, andsecond emission light waveguide 490. Excitation light guide 290, firstemission light waveguide 390, and second emission light waveguide 490are housed in probe 185.

The system's light source is effective in exciting the fluorescentspecies. Suitable light sources include light-emitting diodes, lasers,tungsten lamps, halogen lamps, xenon lamps, arc lamps, and hollowcathode lamps. In one embodiment, the light source is a light-emittingdiode emitting light in the range from 500 to 560 nm. A representativelight-emitting diode useful in the system of the invention is a greenultrabright Cotco 503 series LED commercially available from Marktech,Latham N.Y.

The collimating lens directs light (e.g., excitation light from thelight source or first and second emission light from the emission lightwaveguides) to the bandpass filter. Suitable collimating lenses includeBiconvex glass lenses and Plano-convex glass lenses. Representativecollimating lenses useful in the system of the invention are the TechSpec PCX lenses commercially available from Edmund Optics, Barrington,N.J. The excitation collimating lens is 12×36 (diameter by effectivefocal length in mm) and the first and second emission collimating lensesare 12×18.

The focusing lens focuses light from the bandpass filter to theexcitation light waveguide or from the bandpass filter to the detector.Suitable focusing lenses include Biconvex glass lenses and Plano-convexglass lenses. Representative focusing lenses useful in the system of theinvention are the Tech Spec PCX lenses commercially available fromEdmund Optics, Barrington, N.J. The excitation focusing lens is 12×18and the first and second emission focusing lenses are 12×15.

Filters are used in the optical platform to narrow the bandwidth oftransmitted light.

Suitable excitation filters include bandpass filters, shortpass filters,longpass filters, or a combination of short and long pass filters. Inone embodiment, the system uses a shortpass filter that passes light inthe range from about 370 nm to 540 nm. A representative excitationshortpass filter useful in the system of the invention is 540ASPcommercially available from Omega Optical, Brattleboro, Vt.

Suitable first emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 595 to 605 nm andhas a full width at half height of 10 nm. A representative firstemission bandpass filter useful in the system of the invention is600DF10 commercially available from Omega Optical, Brattleboro, Vt.

Suitable second emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 562 to 573 nm andhas a full width at half height of 10 nm. A representative secondemission bandpass filter useful in the system of the invention is568DF10 commercially available from Omega Optical, Brattleboro, Vt.

The excitation light waveguide transmits excitation light from the lightsource through the probe to the fluorescent species. In one embodiment,the excitation light waveguide includes one or more optical fibers. Inone embodiment, the excitation waveguide is a single optical fiber. Arepresentative fiber optic useful in the system of invention is RO2-534commercially available from Edmund Optics, Barrington, N.J.

The first and second emission light waveguides transmit fluorescentemission from the fluorescent species through the probe to the first andsecond emission detectors, respectively.

In one embodiment, the first emission light waveguide includes one ormore optical fibers. In one embodiment, the first emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thefirst emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system of invention is RO2-533commercially available from Edmund Optics, Barrington, N.J.

In one embodiment, the second emission light waveguide includes one ormore optical fibers. In one embodiment, the second emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thesecond emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system of invention is RO2-533commercially available from Edmund Optics, Barrington, N.J.

Suitable optical fibers useful in the system of the invention includeglass or plastic optical fibers from 0.2 to 2 mm diameter.

The system's first and second emission detectors are effective inmeasuring the first and second fluorescent emissions from thefluorescent species. Suitable detectors include photodiodes, PIN diodes,and photomultiplier tubes. In one embodiment, the first and secondemission detectors are photodiodes responsive in the range from 400 to800 nm. Representative photodiodes useful in the system of the inventioninclude BPW34 commercially available from Vishay Intertechnology,Malvern, Pa.

A representative probe housing excitation and emission light guidesuseful in the system of the invention is illustrated schematically inFIG. 20. As shown in FIG. 20, the light guides are optical fibers.Referring to FIG. 20, probe 185 houses excitation light guide 290, aplurality of first emission light guides 390, and a plurality of secondemission light guides 490. In the representative probe shown in FIG. 20,there are four first emission light guides 390, and four second emissionlight guides 490. The four first emission light guides can be consideredto be a first channel (e.g., measuring the first fluorescent emissionfrom the fluorescent species) and the four second emission light guidescan be considered to be a second channel (e.g., measuring the secondfluorescent emission from the fluorescent species). In the illustratedrepresentative probe, the fibers from each of the two sets of fibersalternate (i.e., alternating fibers 390 and 490) around the centralfiber (290). This configuration provides for evening out of “hot spots”so that light collected by the first set is similar to the lightcollected by the second set.

The relationship between the probe housing the excitation/emission lightguides and the sealed vessel port is illustrated schematically in FIG.21. Referring to FIG. 21, probe 185 is received by port 205. Port 205includes window 210, which is transparent to excitation and emissionwavelengths used in the fluorescent measurement. Excitation lightemanating from light guide 290 passes through window 210 andinterrogates substrate 220 on which the fluorescent species isimmobilized and which, in the operation of the method of the invention,is in contact with the sample contained in sealed vessel 200.Irradiation of substrate 220 results in excitation of thesubstrate-immobilized fluorescent species and fluorescent emission fromthe fluorescent species. Emission from the fluorescent species isreceived by and transmitted through light guides 390 and 490 todetectors 382 and 482, respectively (see FIG. 19B). As noted above, thefluorescent species' first emission intensity and the second emissionintensity will depend on the pH of the sample.

A representative port assembly useful for incorporation into a sealedvessel during manufacture is illustrated in FIG. 22. Referring to FIG.22, port assembly 232 includes port 205 and tip 235. Port 205 is acylinder terminating with window 210 and having opening 212 forreceiving probe 185 (not shown). In one embodiment, port 205 tapers fromopening 212 to window 210 such that the depth of insertion of probe 185into port 205 is predetermined by the probe's diameter. When inserted inthe port, the face of probe 185 and window 210 are substantiallyparallel. Port 205 and tip 235 are adapted such that the port and tipare reversibly connectable. In one embodiment, port 205 includes annularinset 214 and tip 235 includes opening 216 defined by annular lip 218for receiving inset 214. In this embodiment, inset 214 has a diameterless than opening 216. It will be appreciated that the connectingrelationship between the port and tip can be reversed (i.e., port havingannular lip for receiving tip having inset). Lip 218 defines bed 222 forreceiving substrate 220, which is secured in port assembly 202 when port205 is connected to tip 235. Tip 235 includes aperture 224 in bed 222.Aperture 224 provides for contact of substrate 220 with a liquid samplecontained in the sealed vessel.

Fluorescent Species Having pH-Dependent Emission.

In one embodiment, the method and system of the invention for measuringpH uses a fluorescent species having pH-dependent fluorescent emission.The fluorescent species has a first emission intensity at a firstwavelength and a second emission intensity at a second wavelength, thefirst and second emission intensities being characteristic of pH in theenvironment of the fluorescent species. The ratio of the first andsecond emission intensities provides pH measurement. It is appreciatedthat fluorescent emission occurs as a wavelength band having a bandmaximum that is referred to herein as the emission wavelength.

In one embodiment, the separation between the first wavelength and thesecond wavelength is at least about 40 nm. In one embodiment, theseparation between the first wavelength and the second wavelength is atleast about 30 nm. In one embodiment, the separation between the firstwavelength and the second wavelength is at least about 20 nm. Using 10nm HBW filters, the separation is at least about 30 nm. Preferably, thesystem of the invention achieves fluorescence signal separation byremoving any emission band overlap by 10E5 or more.

The method and system of the invention for measuring pH are not limitedto any particular fluorescent species, nor any particular pH range. Themethod and system of the invention is operable with any fluorescentspecies having pH-dependent properties that can be excited and itsemission measured. The range of pH measurable by the method and systemof the invention can be selected and is determined by the pH-dependentproperties of the fluorescent species.

In addition to their pH-dependent properties noted above, suitablefluorescent species include those that can be substantially irreversiblyimmobilized on a substrate. The fluorescent species can be covalentlycoupled to the substrate or non-covalently associated with thesubstrate.

Suitable pH-dependent fluorescent species include those known in theart. Representative fluorescent species having suitable pH-dependentproperties include fluorescein derivatives including naphthofluoresceincompounds, seminaphthofluorescein compounds (e.g., SNAFL compounds), andseminaphthorhodafluor compounds (e.g., SNARF compounds). These compoundshave advantages associated with their long wavelength emission, which isless susceptible to potential interfering light absorbing substances inblood. These compounds also have relatively long wavelength absorbancemaking them particularly suitable for excitation by commerciallyavailable LED light sources. Another compound having suitable pHdependent behavior is HPTS, 8-hydroxy-1,3,6-pyrenetrisulfonic acid.Although the compound has desired ratiometric pH properties, excitationis optimal at short wavelength (403 nm) where strong LED light sourcesare not commercially available. Representative SNAFL and SNARF compoundsuseful in the method and system of the invention are described in U.S.Pat. No. 4,945,171. Molecular Probes (now Invitrogen, Eugene, Oreg.)sells CNF, SNAFL, SNARF fluors with conjugatable carboxylic acid linkergroups, see, for example, Molecular Probes Handbook (Ninth Edition) byR. P. Haugland, Chapter 21 “pH indicators” pages 829-847. EpochBiosciences (now Nanogen, Bothell, Wash.) sells EBIO-3 with a propanoicacid linker. Whitaker et al. (Anal. Biochem. (1991) 194, 330-344) showedthe synthesis of a number of SNAFL compounds. Wolfbeis et al. (MikrochimActa (1992) 108, 133-141) described the use of CNF and aminocelluloseconjugates. The earliest reference to the SNAFL family of compounds isWhitaker et al. (1988) Biophys. J. 53, 197a. A related dye in the CNFfamily is VITABLUE, a sulfonenaphthofluorescein derivative (Lee et al.(1989) Cytometry 10, 151-164) having a pKa of 7.56. A CNF analog withbromine substituents at each carbon adjacent to a phenol (pKa 7.45) hasa pKa that is 0.54 pKa units lower than their measured pKa for CNF (pKa7.99). Lee et al. note that “true” pKa values are difficult to determinefor these compounds. A method for pKa determination is described inExample 12. SNAFL-1 (literature pKa ˜7.8) free acid had a pKa of 7.6 inthat fluorescence-based assay. Other suitable fluorescent speciesinclude the compounds described in U.S. Patent Application PublicationNo. US 2006/0204990 A1, published Sep. 14, 2006 (Ser. No. 11/357,750).

The structures of seminaphthofluorescein compounds (SNAFL-1 and EBIO-3)useful in one embodiment of the method and system of the invention areillustrated below.

The numbering scheme describes position of attachment of linkermolecules. These compounds have carboxylate linking groups suitable forconjugation to carrier proteins, as described below. For conjugation,the reactive N-hydroxysuccinimide (NHS) ester of SNAFL-1 (commerciallyavailable from Molecule Probes, Inc., Eugene, Oreg.) can be used.Conjugation to lysine residues in human serum albumin (HSA) gave desiredSNAFL/HSA conjugates. Carbodiimide activation of EBIO-3 gave a reactiveintermediate that was efficiently conjugated to human serum albumin.

Representative seminaphthofluorescein compounds useful in a method andsystem of the invention are illustrated in FIGS. 23A-23E.

The SNAFL compounds are commercially available from Molecular Probes,Inc., Eugene, Oreg. The SNAFL compounds can be readily synthesizedaccording to general procedures that have been published (see, forexample, U.S. Pat. No. 4,945,171).

The preparation of a representative 2-chloro substituted SNAFL compoundis shown below.

The compound can be prepared by condensation of 1,6-dihydroxynaphthalenewith the diacid substituted 4-acylresorcinol in the presence of adehydrating acid or Lewis acid catalyst, such as zinc chloride.

The preparation of SNAFL compounds having propionic acid linkers isdescribed in U.S. patent application Ser. No. 11/022,039, incorporatedherein by reference in its entirety. A representative SNAFL compoundshaving a propionic acid linker, EBIO-3, is commercially available fromNanogen, Bothell Wash.

The emission spectra as a function of pH of representative fluorescentspecies (i.e., SNAFL-1 and EBIO-1) useful in the method and system ofthe invention are illustrated in FIGS. 24 and 25, respectively. FIG. 24illustrates the emission spectra of SNAFL-1 in 50 mM potassium phosphatebuffer as a function of pH (pH 6.0 to 10.0) (excitation at 540 nm).Referring to FIG. 24, the response at pH 6-7 is relatively poor(pKa=7.6). FIG. 25 illustrates the emission spectra of EBIO-3 in 50 mMpotassium phosphate buffer as a function of pH (pH 6.0 to 10.0)(excitation at 545 nm). Referring to FIG. 25, the response at pH 6-7 isrelatively good (pKa=6.6). Spectral properties and pKa data for theSNAFL analogs illustrated in FIGS. 23A-23E are summarized in Table 1.

TABLE 1 pH-Sensitive absorbance and emission of SNAFL analogs. EmissionAbsorbance Absorbance Emission λmax Compound λmax (acid) λmax (base)λiso (base) pKa SNAFL-1 482, 510 nm 540 nm 585 nm 620 nm 7.6 SNAFL-2485, 514 547 590 630 7.6 EBIO-1 496, 519 545 560 620 6.5 EBIO-2 506, 538572 590 645 7.8 EBIO-3 480, 509 534 560 610 6.6

Referring to Table 1, absorbance and emission spectra were obtained at10 μM SNAFL analog. Absorbance was measured at pH 6, 8, and 10: acid (pH6) gave two bands of similar absorbance; pH 10 gave a single λmax(base). The emission spectra were determined by excitation at theabsorbance λmax (base). The wavelength where emission spectra crossed isreported as λiso. The emission λmax was measured at pH 10. pKa wasdetermined from fluorescence emission spectra. EBIO-1 and EBIO-3 weremore sensitive to changes at pH ˜6.5. The other analogs were moresensitive at pH ˜8.

Fluorescent Species Conjugates for Substrate Immobilization.

For use in a method and system of the invention, the fluorescent speciesmay be immobilized on a substrate such that the fluorescent species isin contact with the sample, the pH of which is to be measured. Thefluorescent species can be immobilized on the substrate through the useof a material (e.g., macromolecular spacer material) having a strongassociative interaction with the substrate. The spacer material allowscovalent conjugation of the fluorescent species and provides largesurface area needed for efficient non-covalent immobilization to thesubstrate surface. In one embodiment, the spacer material is human serumalbumin (HSA) having ˜44 lysine residues available for covalentconjugation. HSA's densely charged molecular structure has a passivatingeffect when adsorbed to biomaterials. Other advantages include reducedfluorescence quenching, uniform environment for the conjugatedfluorophore, and availability in recombinant form (from yeast) so thereis no chance of infection (as with HSA from donors). HSA conjugates areeasily purified by ultrafiltration methods and form stable solutionsthat are easily characterized by absorbance and fluorescence assays todetermine the number of fluorophores per protein.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a protein or protein fragment. Suitableproteins include those that can be substantially irreversiblyimmobilized on the substrate. The protein can be covalently coupled tothe substrate or non-covalently associated with the substrate. Suitableproteins include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the protein.

Suitable proteins include human serum albumin (HSA), bovine serumalbumin (BSA), vonWillebrand's factor, kininogen, fibrinogen, andhemoglobin (no iron). Suitable proteins include proteins havingavailable lysine residues (for conjugation to the fluorophore) andmolecular weight sufficient to allow for immobilization efficiency tothe blot membrane. Other functional groups in the protein (likecysteine) could presumably be used for covalent bonding to suitablyreactive solid supports.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a polysaccharide. Suitable polysaccharidesinclude those that can be substantially irreversibly immobilized on thesubstrate. The polysaccharide can be covalently coupled to the substrateor non-covalently associated with the substrate. Suitablepolysaccharides include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the polysaccharide.

Suitable polysaccharides include dextrans, aminodextrans, heparin, andlectins.

In another embodiment, the fluorescent species is immobilized on thesubstrate through the use of dendrimeric structures. Suitabledendrimeric structures include those that can be substantiallyirreversibly immobilized on the substrate. The dendrimeric structurescan be covalently coupled to the substrate or non-covalently associatedwith the substrate. PAMAM dendrimers are commercially available as areother structural types and sizes.

In one embodiment, the fluorescent species is covalently coupled to aprotein to provide a fluorophore-protein conjugate that can beimmobilized on a substrate. In one embodiment, thefluorophore-polysaccharide conjugate is non-covalently associated withthe substrate.

In one embodiment, a fluorophore-protein conjugate is immobilized on asubstrate. In one embodiment, the fluorescent species is aseminaphthofluorescein and the protein is human serum albumin. In oneembodiment, the seminaphthofluorescein is SNAFL-1. The preparation ofSNAFL-1/HSA conjugates is described in Example 13. The fluorescentproperties of SNAFL-1/HSA conjugates are described in Example 14. In oneembodiment, the seminaphthofluorescein is EBIO-3. The preparation ofEBIO-3/HSA conjugates is described in Example 1. A schematicillustration of the coupling of EBIO-3 to HSA is illustrated in FIG. 26.The fluorescent properties of EBIO-3/HSA conjugates are described inExample 15.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (SNAFL-1/HSA, 1.6fluorophores per HSA) useful in the method and system of the inventionare illustrated in FIG. 27.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (EBIO-3/HSA, 1.92fluorophores per HSA) useful in the method and system of the inventionare illustrated in FIG. 28.

For the fluorophore-protein conjugate, the optimum fluorophore loadingwill vary depending on the particular fluorophore.

For SNAFL-1/HSA conjugates the fluorophore loading can vary from about0.01 to about 38 SNAFL-1/HSA. Low signal at 0.01 and fluorescentquenching at 40 fluorophores/HSA. In one embodiment, the SNAFL-1conjugate includes about 2 SNAFL-1/HSA.

For EBIO-3/HSA conjugates the fluorophore loading can vary from about0.01 to about 40 EBIO-3/HSA. In one embodiment, the EBIO-3 conjugateincludes about 2 EBIO-3/HSA.

Substrates for Fluorescent Species Immobilization.

In the method and system of the invention, the fluorescent species isimmobilized on a substrate. As noted above, the fluorescent species canbe directly immobilized on the substrate covalently or by non-covalentassociation or, alternatively, through the use of a material (e.g.,fluorophore-protein conjugate) that can be immobilized on the substratecovalently or by non-covalent association.

Suitable substrates substantially irreversible immobilized thefluorescent species. In the method of the invention, suitable substratesalso do not inhibit the contact of the liquid sample with thefluorescent species and do not impair or alter the pH measurement.

Representative substrates include membranes, such as microporousmembranes made of cellulose, nitrocellulose, mixed esters ofnitrocellulose and cellulose acetate, polyethylene terephthalate,polycarbonate, polyvinylidene fluoride and polyimide. Such materials areavailable commercially from Whatman S&S, Florham Park, N.J. andMillipore, Billerica Mass. Suitable membranes include membranes in whichthe microporous structure is created by ion beam penetration such asmembranes commercially available from Oxyphen Gmbh, Dresden, Germanyunder the designation OXYPHEN. Charged nylon surfaces (Nytran) can alsobe used. Suitable membranes include plastic membranes in which themicroporous structure is made by injection molding the micropores intothe plastic such as the processes used by Amic, Stockholm, Sweden.Emission intensity of SNAFL-1/HSA at pH 7 immobilized on various poresize mixed ester nitrocellulose cellulose acetate membranes is shown inFIG. 38.

Immobilization of representative fluorophore protein conjugates onmembranes is described in Examples 16 and 2. Example 16 describes theimmobilization of SNAFL-1/HSA conjugates. Example 17 describes thefluorescent properties of immobilized SNAFL-1/HSA conjugates. Example 2describes the immobilization of EBIO-3/HSA conjugates. Example 18describes the fluorescent properties of immobilized EBIO-3/HSAconjugates.

The emission spectra of a representative fluorophore-protein conjugate(SNAFL-1/HSA, 3.6:1) immobilized on Oxyphen and nitrocellulose as afunction of pH (pH response), as measured by the microwell assaydescribed in Example 17, are illustrated in FIGS. 29 and 30,respectively.

The emission spectra of a representative fluorophore-protein conjugate(EBIO-3/HSA, 2.0:1) immobilized on nitrocellulose, as described inExample 16, as a function of pH (6.0, 6.5, 7.0, 7.5, 8.0, and 10.0), asmeasured by the telescoping tube insert assay described in Example 18,are illustrated in FIG. 31. The large spread of emissions at 600 nm forthe pH 6 to 8 range indicates good fluorescence verses pH response.

Ratiometric pH Method and System.

In one embodiment, the method of the invention is a fluorescentwavelength-ratiometric method. In the method, the first and secondfluorescent emission intensities of the fluorescent species measured atfirst and second emission wavelengths, respectively, are ratioed toprovide pH information. The first emission wavelength varies with pHwhile the second emission wavelength is constant with pH and gives aninternal control for the fluorescent intensity. In one embodiment, alookup table is used to lookup a combination of the measured ratio,first and second emission wavelength and determines its correspondingpH. In one embodiment, a mathematical function of the ratio, first andsecond emission wavelength is used to calculate the resulting pH. FIG.15 is a lookup table that correlates the ratio of the two emissionintensities to pH. FIG. 16 is an example of data from calibrationstudies that are used to generate a lookup table such as the one in FIG.15.

FIG. 32 illustrates the data used in the method of the invention formeasuring pH. The emission spectra of a representativefluorophore-protein conjugate (EBIO-3/HSA, 2:1) immobilized onnitrocellulose at pH 7.0 is shown as measured by the telescoping tubinginsert assay. In this setup, the excitation bandpass filter was unableto completely remove the excitation light in the emission region asillustrated by the background signal measured on a blank nitrocellulosedisc. The full spectrum corrected for the background was multiplied bythe transmittance of the first and second hypothetical filters at eachwavelength and the area under the resultant curve was calculated to givea signal for the first and second wavelength. The center wavelengths andbandwidths of hypothetical filters were chosen such that the ratiometricproperties of the conjugate had the strongest relationship to the pH inthe region of interest.

FIG. 33 illustrates the results of the method of the invention forphosphate buffered saline (PBS), platelet poor plasma (PPP), andplatelet rich plasma (PRP) as measured by the telescoping tubing insertassay described in Example 18. The three curves represent the best fitrelationship between the measured pH and ratios for the three differentliquids.

FIG. 34 illustrates the correlation of pH results for three differentplasma samples obtained by the method and system of the invention asmeasured by the injection molded insert PVC tube assay described inExample 18. The relationship between the fluorescent signal and the pHhas an accuracy of about 0.1 pH units.

FIG. 35 illustrates stability of a representative substrate-immobilizedfluorophore conjugate of the invention (EBIO-3/HSA, 2:1) on mixed esternitrocellulose and cellulose acetate prepared by the soaking method andas measured by the leaching assay described in Example 2. The low levelof leaching is far below the toxic level for any compound.

Carbon Dioxide Measurement.

In another aspect, the present invention provides a device and methodfor measuring carbon dioxide concentration in a liquid sample. Thecarbon dioxide measuring method utilizes the pH measuring method andsystem described above. In the carbon dioxide measuring method anddevice, a substrate-immobilized fluorescent species as described aboveis in contact with a solution, the pH of which is responsive to carbondioxide level. In addition to being in contact with thesubstrate-immobilized fluorescent species, the solution having pHresponsive to carbon dioxide level is in contact with a liquid samplefor which the level of carbon dioxide is to be measured. The solutionhaving pH responsive to carbon dioxide level is isolated from the liquidsample for which the level of carbon dioxide is to be measured by aselectively permeable membrane. The membrane is permeable to gases(e.g., carbon dioxide) and impermeable to other materials (e.g.,liquids). Using the method of measuring pH described above, the pH ofthe solution responsive to carbon dioxide concentration in contact withthe substrate-immobilized fluorescent species is measured and correlatedwith the carbon dioxide level of the sample in contact with thatsolution.

The solution having pH response to carbon dioxide level is an aqueoussolution that includes an agent that is reactive toward carbon dioxideand changes the pH of the solution in response to carbon dioxideconcentration. Suitable agents that are reactive toward carbon dioxideand change the pH of the solution in which they are dissolved includebicarbonates, such as sodium bicarbonate.

The selectively permeable membrane isolates the solution having pHresponsive to carbon dioxide level from the liquid sample containingcarbon dioxide. The membrane is permeable to carbon dioxide andimpermeable to liquids and other solutes. In the method, carbon dioxidefrom the liquid sample passes from the liquid sample through themembrane and into the aqueous solution thereby reacting with the carbondioxide reactive agent to alter the pH of the aqueous solution. Suitableselectively permeable membranes include membranes made from silicone andPTFE.

FIG. 36 illustrates a representative device of the invention formeasuring carbon dioxide in a sealed vessel. Referring to FIG. 36,device 600 includes port assembly 610 including port 620 and tip 630.Port 620 is a cylinder terminating with window 622 and having opening624 for receiving probe 185. When inserted in the port, the face ofprobe 185 and window 622 are substantially parallel. Port 620 and tip630 are adapted such that the port and tip are reversibly connectable.Substrate 640 including immobilized fluorescent species is securedwithin port 620 and tip 630. Tip 630 includes a chamber 645 forreceiving a solution having pH responsiveness to carbon dioxide. Chamber645 is defined by window 622, tip 630, and selectively permeablemembrane 650. Chamber 645 includes substrate 640, which is interrogatedby probe 185.

A device for measuring carbon dioxide was assembled as described abovewith the membrane containing immobilized EBIO-3/rHSA conjugate (rHSA isrecombinant HSA). A layer of PARAFILM M, a blend of olefin-typematerials, was added under the membrane towards the tip. The membranewas hydrated with 5 ul of 35 mM carbonate buffer (pH 7.4), which wassealed within the assembly by the PARAFILM M and remained hydratedthroughout the assay. The assembly was subjected to 100% carbon dioxidegas by connection to the gas source with tubing and a “Y” adapter tobleed off the pressure. The assembly was subjected to the carbon dioxidefor an allotted period of time, allowed to acclimate to ambient airconditions, and repeated. The fluorescence was measured at each stage at568 nm and 600 nm after being excited at 525 nm. The results aresummarized below in Table 2 and reflect changes in fluorescence due tothe change in carbon dioxide concentration demonstrating that thefluorometric ratio method of the invention can also be used to calculatecarbon dioxide concentration. The PVC storage bags that are used forplatelet storage are somewhat gas permeable, and carbon dioxide isdirectly related to the measurement of pH.

TABLE 2 Carbon dioxide sensing results. Emission at Emission atEnvironmental Conditions 568 nm 600 nm Ratio (600/568) 15 min. atambient CO₂ 753 2184 2.9 5 min. at 100% CO₂ 1179 2234 1.894 15 min. atambient CO₂ 833 2175 2.611 8 min. at 100% CO₂ 1161 1930 1.662 60 min. atambient CO₂ 765 2184 2.854

The present invention provides a fluorescence-based pH indicator thatcan be easily inserted into the sampling ports of designed blood storagebags and interrogated using a fiber optic-based LED light source andphotodiode measurement system. In one embodiment, this solid statesystem uses a “ratiometric” calibration method that accounts forvariability in fluorescent signal strength due to interfering substancesin blood that may interfere with the amount of excitation light thathits the indicator dye. The ratio of fluorescence intensities aremeasured at two wavelengths, one to detect the acid (protonated) isomerof the dye and one to detect the base (deprotonated) isomer.

To develop an accurate pH detector for platelet rich plasma, compoundshaving pKa of ˜6.6 are suitable, for example, 2-chloro substitution ofSNAFL compound lowers the pKa of the phenol from 7.6 to ˜6.6. Conjugatesof these compounds can be immobilized to various solid supports toprovide sensing pH membranes.

The present invention provides an inexpensive, easy to manufacture pHsensing membrane that gives accurate measurement of pH in plateletstorage bags at pH 6.5-7.5. In one embodiment, the invention uses aprotein conjugate (human serum albumin) of a 2-chloro substitutedratiometric fluorescent compound. The fluorophore:HSA ratio wasoptimized for performance when immobilized to a nitrocellulose blotmembrane. After drying on the membrane, the fluorophore:HSA conjugatehas very low leaching rates. Discs of this material are easily assembledinto holders for insertion into the sampling ports of platelet storagebags. The fluorescent membrane materials showed good pH response using agreen LED based fluorometer. In the method, two emission wavelengths forratiometric pH detection are measured with properly filtered photodiodeswith an accuracy of ˜0.1 units at the desired low pH threshold of 6.5.

Fluorescent probe molecules can be designed to be sensitive to a varietyof environments. The method and system of the invention describes theuse of pH-sensitive fluorophores. However, other environments can beinterrogated by the method and system of the invention modified toinclude environment-sensitive fluorophores other than pH-sensitivefluorophores. A variety of fluorescent probes that change fluorescentproperties as the molecular environment changes are commerciallyavailable. See, for example, Molecular Probes Handbook (9^(th) Ed.) byR. P. Haugland. Probes can be linked to albumins or other proteins andused to prepare substrates for interrogation as described in herein orusing other fluorescent-based methods. Examples of environment-sensitivefluorophores, systems, and methods include the following.

Nucleic acid detection: nucleic acid binding dyes change fluorescentproperties in the presence of DNA or RNA.

Enzyme substrates: proteins or peptides can be labeled with fluorescentdyes and fluorescent quenching molecules such that a fluorescent signalis generated in the presence of particular enzymes such as proteases(FRET detection).

Probes for lipids: lipophilic dyes can change fluorescent properties inthe presence of cell membranes or other lipid rich analytes.

Probes for oxygen: in addition to pH detection and carbon dioxidedetection, certain fluorescent molecules are sensitive to changes inoxygen concentration, for example, tris(2,2′-bipyridiyl)ruthenium(II)dichloride (RTDP).

Indicators for metal ions: fluorescent dyes that bind metals can changefluorescent properties upon binding calcium, magnesium, zinc, sodium,potassium, among other.

Glucose detection: certain lectins such as ConA bind glucose, andsuitably labeled lectins can be prepared as probes for glucose.

One object of the invention is to at least measure the parametersdescribed above in order to solve some of the problems associated withplatelet storage in sealed sterile containers. There are severalproblems or disadvantages described hereinafter that may be solved bythe current invention as multiple parameters are monitored over timewithin the environment of the sealed platelet storage device. Theadditional advantages, objects, or computational features of theinvention described herein will become apparent to those having someskill in the art upon examination or practice of the invention. Theadvantages and objects of the invention may be attained as particularlypointed out in the appended claims.

The following examples are provided for the purposes of illustrating,not limiting, the invention.

EXAMPLES Example 1 The Preparation of Representative Fluorophore-ProteinConjugates: EBIO-3/HSA

Method A.

A 0.1 M stock solution of EDC (Sigma/Aldrich Chemical Co., St. LouisMo.) was prepared by dissolving 6.2 mg of EDC in 0.2 mL of DMF and 0.123mL of 50 mM phosphate buffer (pH 5.8). 1.0 mg of EBIO-3 acid (Nanogen,Bothell, Wash.) was dissolved in 0.102 mL of DMF to give a 20 mMsolution. 3.0 mg (0.045 micromoles) of HSA (Sigma/Aldrich Chemical Co.,St. Louis, Mo.) was dissolved in 0.3 mL of pH 8.5 sodium bicarbonate ineach of two 1.7 mL Eppendorf tubes. 0.1M EDC (0.045 mL) was added to 20mM EBIO-3 (0.045 mL, 0.9 micromoles) in a separate Eppendorf tube andthis was added to one of the HSA tubes to give an EBIO-3:HSA offeringratio of 20:1. An offering ratio of 5:1 was used in the other HSA tubeby adding a premixed solution of 0.0225 mL of EDC (0.1 mM) and 0.0225 mLof EBIO-3 (20 mM). The homogeneous dark red HSA conjugate solutions wereincubated at room temperature in the dark. After 21 hours, each of theHSA conjugates was purified on a G15 Sephadex column as described abovefor the SNAFL conjugates (Example 13). Some unreacted EBIO-3 acidremained at the top of the column (especially for the 20:1 offeringratio), but was cleanly separated from the desired protein conjugatethat eluted first as a pink fraction in ˜0.5 mL of pH 7.4 buffer. Eachof the purified conjugates was diluted to 0.75 mL with pH 7.4 PBS togive 4 mg/mL solutions (0.06 mM). The red solutions were storedrefrigerated and protected from light. 1 micromolar solutions of eachEBIO-3/HSA conjugate were prepared at pH 7.4 and analyzed by UV-visspectra using a Beckman DU640B spectrometer. The free EBIO-3 acid (10micromolar) spectrum had absorbance maximum at 534 nm, the 20:1conjugate had absorbance at 538 nm and the 5:1 conjugate had maximum at545 nm. The spectra showed the expected increase in absorbance withincreasing EBIO-3:HSA offering ratio. Using this EBIO-3 acid as astandard, the 20:1 conjugate had 5.07 EBIO-3:HSA and the 5:1 offeringhad 1.92 EBIO-3:HSA. The coupling efficiency was somewhat lower than forthe SNAFL/HSA conjugates of Example 4 (the 20:1 conjugate had 11.2fluors/HSA and the 5:1 offering had 4.1 fluors/HAS). The EDC couplingmethod was suitably efficient and reproducible.

Method B.

A 0.1 M solution of EDC (Sigma/Aldrich Chemical Co., St. Louis, Mo.) isprepared by dissolving 6.0 mg of EDC in 0.194 mL of DMF and 0.118 mL of50 mM PBS (pH 7.4). 3.0 mg of EBIO-3 acid (Nanogen, Bothell, Wash.) isdissolved in 0.306 mL of DMF to give a 20 mM solution. The two solutionsare combined in the EBIO-3 solution container and incubated at roomtemperature for one hour in the dark.

75.0 mg (1 micromole) of liquid recombinant HSA (rHSA) from yeast (DeltaBiotechnology, Ltd., Nottingham, UK) is mixed with 7.5 mL of pH 8.5sodium bicarbonate in a 15 mL conical tube. The entire contents of theEBIO-3/EDC solution are combined with the rHSA solution and incubated atroom temperature in the dark for 15-20 hours. The rHSA/EBIO-3 conjugateis purified using the Amicon stirred ultrafiltration cell system and aYM10 membrane (Millipore, Bedford, Mass.). A 50 mM PBS (pH 7.4) is usedas the wash solution. After purification, the protein concentration ofthe conjugate is measured using the BCA™ Protein Assay (Pierce,Rockford, Ill.). An aliquot of the conjugate is diluted to 1 mg/mL with50 mM PBS (pH 7.4) based on its BCA determined protein concentration.The 1 mg/mL aliquot of conjugate, the last milliliter of PBS effluent,an aliquot of the 50 mM PBS (pH 7.4), and an aliquot of the EB3 Standard(15 mM EBIO-3 solution in DMF and 50 mM PBS (pH 7.4)) are analyzed viaan absorbance scan utilizing Bio-Tek's Synergy HT plate reader. The scanis taken on 300 microliters of each of the above mentioned samples in ablack, 96-well, clear, flat bottom plate, scanned from 450 nm to 650 nm.Their max peaks are recorded and used to determine purity and quality ofthe conjugate.

Example 2 Immobilization of Representative Fluorophore-ProteinConjugates: EBIO-3/HSA

Spotting Immobilization Method.

EBIO-3/HSA conjugate was prepared as described in Example 1 at a ratioof 2:1. Nitrocellulose membranes were obtained from Schleicher andSchuell under the trade name PROTRAN. The discs were treated in the sameway as the general immobilization method described in Example 14 using a4 mg/mL solution of EBIO-3/HSA.

Soaking Immobilization Method.

EBIO-3/HSA conjugate was prepared as described in Example 1 at a ratioof 2:1. Mixed ester nitrocellulose and cellulose acetate membranes wereobtained from Millipore under the product series TF. The EBIO-3/HSAconjugate is diluted to 0.2 mg/mL and 45 mL is added to a 9 cm disc ofthe membrane. The disc is agitated overnight at room temperature andprotected from light. The unbound conjugate is removed and the disc iswashed with two 1 hour washes and one overnight wash all with agitation.The disc is then desiccated and stored dry. Smaller discs are punchedfrom the 9 cm disc for studies.

Example 3 The Manufacture of a Vessel Incorporating a RepresentativeSubstrate-Immobilized Fluorescent Species

PVC material is compounded with a number of additives, for example,plasticizers, stabilizers, and lubricants. The formulation is used formaking bags and tubes. The compounded PVC is extruded through a die orcalendared in a press for converting the plasticized material into sheetform. The extruded sheet, after slitting, is cut into the desired sizeand sent to the welding section. The donor and transfer tubings are madeby extrusion of similar PVC compounds. The tubes are then cut to theappropriate length and sent to the welding section. The components, suchas transfusion ports, needle covers, and clamp, are produced byinjection molding. The components are ultrasonically cleaned and driedin a drying oven.

Welding.

The blood bags are fabricated by a high frequency welding technique.Sized PVC sheets are placed between electrodes and high frequency athigh voltage is applied. PVC gets heated very rapidly and sealing takesplace between electrodes. Transfusion ports and donor and transfertubing are kept in the appropriate position with the bag and welded toform an integral part of the blood bag system. For the manufacture of avessel incorporating a representative substrate-immobilized fluorescentspecies, an open tube is welded to provide port 510A. The tube can bemade of colored PVC to provide light protection for the immobilizedfluorescent species. Welded bags are trimmed. The port assembly 232(FIG. 22) is manufactured from injection molded Lexan parts (205 and235) and a 3.53 mm ( 9/64 inch) diameter nitrocellulose disc withimmobilized fluorescent species (220). The port assembly is heldtogether by friction fit or can be glued in place. The port assembly isinserted in the open tube of port 510A. The port assembly is held in theport by friction fit or can be glued in place. The assembled bag andport assembly is sterilized and labeled for ultimate storage of plateletconcentrates.

Example 4 The Incorporation of a Representative Substrate-ImmobilizedFluorescent Species into a Sealed Vessel

The port assembly is manufactured from injection molded Lexan parts anda 3.53 mm ( 9/64 inch) diameter nitrocellulose disc with immobilizedfluorescent species. The port assembly is held together by friction fitor can be glued in place. The port assembly is inserted through theseptum seal inside port 510A by puncturing the seal with the spiked tip.Alternatively, the seal can be pre-punctured with a separate spike tool.The insertion of the port assembly can be performed on either empty orplatelet filled bags, but in either case, aseptic methods should be usedto avoid possible contamination of the bag contents. The port assemblyis held in the port by friction fit or can be glued in place. The vesselremains sealed (leakproof) after insertion of the port assembly in theport.

Example 5 Assembly of Inserts and Bags

The methods described in Examples 3 and 4, above, were followed with thefollowing exceptions: an altered version of the injection molded inserttip 235, shown in FIG. 22, was used. After friction fit assembly, theshaft, tip and membrane disc were pushed into an opaque blue sleeve ofPVC tubing (Natvar, City of Industry, California, durometer 80 Shore A,dimensions=ID 0.216″, OD 0.291″, L 1.15″) until the flange contacted thetubing. The prongs just reach the other end of the tubing. This frictionfit assembly was fit inside the assembled large bag as shown in FIG. 1.

Assembly of Inserts into Bags.

The bags were welded with large diameter PVC tubing (inner diameterslightly smaller than the outer diameter of the blue insert assembly, ¼inch shorter than the blue insert assembly). The blue insert assemblywas held into the larger diameter PVC tube in the bag by friction fit orby solvent welding with cyclohexanone. The blue sleeve protrudes fromthe flush cut bag tubing by ¼ inch, thus providing a space for the“clip” on the instrument.

Preparation of Small Bags.

Method A.

The small bag shape shown in FIG. 2 was welded from previously assembledlarge bags (with no inserts or twist off sample ports). One of thetubing ports for the sample ports is discarded along with much of thecitrate PVC film. The blue pH reading insert assembly, pigtail tubingand sample ports were carefully assembled with cyclohexanone solventwelding and packaged for sterilization.

Method B.

Two sheets of breathable citrate PVC film (Solvey-Draka) are RF weldedtogether using a steel tool that contacts the film in the outer shape ofthe bag in FIG. 2. PVC tubes on steel rods are also placed between thetwo layers of the film before the RF welding. This forms a leak proofseal with the tubes and film. After the RF welding the steel rods areremoved so that twist off ports, pigtails and sensors can be placed inthe PVC tubes. These features are solvent bonded or fixed in another wayinto the tubes.

Ethylene Oxide Sterilization of Bag/Insert Assembly.

The bags were individually wrapped and sterilized using a standardethylene oxide cycle used for other platelet storage bags. Theoverwrapped bags were packaged in boxes and unwrapped just prior to use.

Example 6 Protocol Description for Large Bag pH and Platelet Health

An in vitro evaluation of PCs during storage in representative large andsmall platelet storage containers with integrated pH probes wasperformed. The evaluation determined that the integrated pH device hadno negative impacts on in vitro quality of platelets during storage,either during storage in standard platelet containers or during storagein down-scaled containers for research purposes. The following examplerefers to the data presented in FIGS. 3-7.

Platelet Storage Containers

A. 4 large experimental PVC-citrate storage containers (lot numberm4340) with integrated pH probe (for storage of about 300 mL PC), ETOsterilized.

B. 4 large approved PVC-citrate storage containers (brand Fresenius,T2110, batch H10211011) (for storage of about 300 mL PC), steamsterilized.

C. 4 small experimental PVC-citrate containers with integrated pH probe(for storage of about 17 mL PC), ETO sterilized.

D. 4 small PVC-DEHP (so-called buffy coat bags; 100-150 mL nominalvolume), steam sterilized.

All other materials are standard materials used by the Sanquin BloodCentre Region North West for blood collection and by the SR-BTTlaboratory for blood processing and testing.

A representative system of the invention (serial number PH05100007,firmware version 2.00, Blood Cell Storage, Inc., Seattle, Wash.) wasused for non-invasive fluorescence measurements of the storagecontainers with integrated pH probes. For the data presented in FIGS.3-10 and 16, each bag was positioned on a representative system (device)of the invention and the fluorescent ratio was determined from which pHwas calculated. Readings/samplings were done at 4 hours post set-up thenevery 6-12 hours later over a period of 7 to 11 days. The volume in thesmall storage containers was to about 15 mL.

The first two PC were combined, and then two small bags were filled witheach about 15 mL, followed by distribution of the remaining volume intwo equal volumes over the two types of large storage containers.

In order to ascertain a good starting quality of the platelets, ameasurement of CD62P expression on day 1 of storage was included.

The number of platelets positive for PAC-1 (fibrinogen binding site onglycoprotein IIb/IIIa) was not determined, because the test was shown tobe influenced by the presence of plasma.

Blood Collection

Standard blood collection systems (manufactured by Fresenius HemoCare,Emmer-Compascuum, the Netherlands) were filled with approximately 500 mLof blood during blood collection at the Sanquin Blood Centre RegionNorthWest (Sanquin Blood Supply Foundation, Amsterdam, the Netherlands)from non-enumerated, informed donors. The day of blood collection wasdesignated as day 0 of the study.

Blood collections were performed under standard conditions, with the aidof calibrated blood collection balances equipped with a mixing platformallowing mixing at regular intervals, monitoring of blood flow andbleeding time, a final check of the weight of the donation and coolingof the blood to 20° C. immediately after collection. At least 40donations (20 from A-Rh(+) and 20 from 0-Rh(+) donors) meeting thecriteria of volume (500±50 mL of blood) and bleeding time (<15 min) wereselected by the Blood Centre for further processing to plasma, red cellconcentrate in SAG-Mannitol and buffy coat 12 to 16 h after the bloodcollection (on day 1 of the study). After centrifugation and Compomat G4separation of the whole blood, at least 40 buffy coats were delivered tothe BTT laboratory. In addition, per set of 5 buffy coats a plasma unitfrom one of the corresponding 5 donations was delivered.

Preparation of Pooled Platelet Concentrates

The 40 buffy coats were used to prepare 6 pools, each consisting of 5buffy coats and one unit of plasma. Pools consist of units of the sameblood group. From these buffy coat pools, platelet concentrates wereprepared after a second centrifugation step (Hettich Roto Silenta/RP, 5min at 1700 rpm, 905×g, brake 3). Conditions for these separations areselected in such a way that from each pool of buffy coats a plateletconcentrate (PC) of ±320 mL volume with 1-1.5×10e9 platelets/mL and aleukocyte contamination of less than 50×10e7 leukocytes per concentratewas obtained. During preparation of PC from the pooled buffy coats, theconcentrates are filtered over a Compostop CS leukoreduction filter(Fresenius Hemocare, 50 cm², T3995) with a large PVC-citrate containerconnected to the outlet of the filter. For 4 PC this container wascontainer A and for 4 PC this was container B (see Materials andInstruments; A and B were matched for blood group).

Pooling and Splicing of Platelet Concentrates

After preparation, a PC in container A was combined with a PC incontainer B (blood group matched) and after mixing two times 15 mL wastransferred to a container C and a container D. Subsequently, theremaining volume was redistributed over the A and B containers in such away that the volume was equal.

Storage of Platelet Concentrates

The filtered PC were stored for 8 days after preparation at 22° C.±2° C.horizontally shaking with 1 cycle per minute. Samples were taken underaseptic conditions after 1, 2, 3, 6, 7 and 8 days of storage (meaningday 2, 3, 4, 7, 8 and 9 of PC shelf-life) and analyzed for variousplatelet quality parameters.

Measurements

The following features of platelet quality were measured:

1. Morphological parameters (measured after 3 days, 6 days and 8 days ofstorage). The morphological parameters involve the screening of theswirling (resulting in a swirling score) and the microscopiccharacterization of the different forms of platelets, i.e. discoid,discoid with dendrites, balloons or spheres, resulting in a so-calledKunicki score.

2. Physical changes (measured after preparation and after 1, 2, 3, 6, 7and 8 days). The physical changes are characterized by measurements ofthe pH, P(O₂) and P(CO₂) and the number of leukocytes (counted at day 0)and the number of platelets.

3. Changes in activation degree (measured after 3 days, 6 days and 8days). The changes in degree of activation were measured with amonoclonal antibody, directed against activation-dependent antigenCD62P. Also the percentage of platelets expressing phosphatidyl-serine(PS, measured with AnnexinV) was determined.

4. Metabolic changes (measured after 3 days, 6 days and 8 days). Themetabolic changes are characterized by the intracellular amount ofadenine nucleotides, the extracellular concentration of glucose andlactate and the mitochondrial membrane potential (as measured withJC-1).

At the end of the storage period, the platelet concentrates in the largecontainers were checked for sterility.

A. Composition of the Platelet Concentrates.

During preparation of the platelet concentrates from the pooled buffycoats, no deviations from normal procedures were observed, with afiltration time over the in-line filter of about 6 min. The eightleukodepleted products were used to prepare 4 pools, which weresubsequently distributed over the small containers (type C and D, each15 mL) and the large containers (type A and B, about 300 mL each). Table2 shows that the PC in type A and type B containers were very similar.After leukoreduction, the number of leukocytes was well below 1×10⁶ inall units (as determined by fluorescent Nageotte counting, with forevery sample 0 counted). The total number of platelets in each poolexceeded 300×10⁹, and these numbers meet the requirements of the Councilof Europe for leukocyte depleted PC.

TABLE 3 Composition of the platelet concentrates Type A Type B Volume PCmL 313 ± 5.8 308 ± 6.2 Platelet conc. ×10⁹/mL 1172 ± 58.0 1172 ± 58.0Total platelets ×10⁹ 367 ± 16.8 361 ± 18.3 Total WBC ×10⁶ 0.10 ± 0.080.09 ± 0.07Values given are the mean±SD of 4 concentrates.

B. Change in morphological parameters.

The viability of platelets after transfusion correlates fairly well withplatelet morphology. The most discoid platelets have the best in vivosurvival, whereas spherical platelets perform much less. A morphologicalindex was introduced by Kunicki to predict viability of platelets aftertransfusion. The number of discs identified in a 100 cell count of fixedplatelets under the microscope is multiplied by 4, spheres by 2,platelets with dendrites by 1, and balloons by 0, resulting in a maximalscore of 400 for perfect discoid platelets.

The morphology scores and the percentage discoid platelets duringstorage of the PC pools are depicted in Table 2.

TABLE 4 MORPHOLOGICAL PARAMETERS OF PLATELET CONCENTRATES DURING STORAGEDay 4 of Day 7 of shelf-life shelf-life Day 9 of shelf-life Bag Kunicki% Kunicki % Kunicki type score discoid score discoid score % discoid AMean 295 50 279 44 248 31 SD 20.4 8.2 11.8 4.8 8.7 2.5 B Mean 288 46 26338 228 24 SD 22.5 9.5 15.5 6.5 8.7 2.5 C Mean nd nd 268 39 nd nd SD ndnd 5.0 2.5 nd nd D Mean nd nd 263 36 Nd nd SD nd nd 17.1 8.5 Nd ndValues given are the mean±SD of 4 concentrates. Days indicate the numberof days after blood collection (day 0). nd: not determined.

The first measurement, after 3 days of storage (day 4 of PC shelf-lifeindicated in the table), indicates a high quality of the platelets.During further storage the morphology score remains high, with onlyminimal differences between the two types of large containers tested.Also the values for the small containers, only determined on day 7, werevery similar, indicating also good storage characteristics for thesesmall containers.

Another method to judge morphology of platelets is the observation ofthe swirling pattern in the storage bag. The “silk-like” patterns areobserved visually, after brief squeezing of the bag and the degree ofinhomogeneity is scored according to the following scale:

3. Swirling inhomogeneity throughout the whole bag, with contrastobservable as fine detail

2. Swirling inhomogeneity visible throughout the whole bag with goodcontrast

1. Some inhomogeneity visible, but only in a few places and with poorcontrast

0. Turbid homogeneity, no effect of squeezing.

With this method for freshly prepared platelet concentrates a score isobtained of 3, whereas after storage for 6 days the score should beabove 2.

Table 2A shows that during storage for up to 8 days (day 9 ofshelf-life) the score in the large containers was above 2, with minimaldifferences between the two tested types of container. In the smallcontainers the score was above 2 till day 6, whereas the swirling scorewas slightly lower in the small PVC-DEHP bag compared to the smallPVC-citrate bag.

TABLE 5 Swirling scores of platelet concentrates during storage Type day2 day 4 day 7 day 8 day 9 Mean A 3.0 2.6 2.5 2.4 2.1 SD 0.0 0.1 0.0 0.30.3 Mean B 3.0 2.8 2.2 2.2 2.3 SD 0.0 0.2 0.3 0.1 0.2 Mean C 3.0 2.8 2.42.4 2.2 SD 3.0 0.2 0.1 0.1 0.1 Mean D 3.0 2.9 2.0 1.9 1.6 SD 3.0 0.1 0.20.1 0.1Values given are the mean±SD of 4 concentrates. Days indicate the numberof days after blood collection (day 0). nd: not determined.

C. Change in Physical Parameters.

When during storage of platelets the pH falls below 6.7 (measured at 37°C.), disc-to-sphere transformation occurs and morphology of theplatelets becomes worse. This change becomes irreversible when the pHfalls below 6.5 and therefore, at the end of storage, CE requirementsprescribe a pH between 6.6 and 7.2 (measured at 37° C.) or between 6.8and 7.4 (measured at 22° C.). The pH values during storage in the largecontainers type A and B and the values for pCO₂, pO₂ and bicarbonate aregiven in Table 3a. During storage the pH remains above 6.9 during 8 days(=9 days of shelf-life), with only minimal differences between the twocontainer types tested. In the small containers (type C and D) the pHwas only measured on day 6 of storage. Compared to the large containersthe pH was slightly lower on this day, indicating slightly worse storageconditions in the small containers. pCO₂ and pO₂ showed the normalpattern during storage of platelet concentrates.

TABLE 6 Physical parameters during storage pH at 37° C. pCO₂ pO₂ HCO₃ ⁻Shelf-life type mean SD Mean SD mean SD mean SD Day 1 A 7.058 0.023 66.52.1 156.0 2.7 18.3 0.5 B 7.058 0.023 66.5 2.1 156.0 2.7 18.3 0.5 Day 2 A7.196 0.013 43.8 0.5 80.6 6.6 16.6 0.5 B 7.173 0.008 46.7 0.5 65.8 10.516.8 0.4 Day 3 A 7.229 0.019 36.5 1.8 87.4 7.3 14.9 0.7 B 7.200 0.00639.1 1.6 73.2 4.7 14.9 0.7 Day 4 A 7.229 0.010 33.4 0.6 84.3 8.8 13.60.4 B 7.190 0.008 36.2 2.1 65.6 5.2 13.5 0.6 Day 7 A 7.095 0.018 30.21.5 98.4 6.1 9.1 0.7 B 7.077 0.010 32.1 1.7 84.6 5.6 9.2 0.3 Day 7 C7.052 0.089 19.5 1.3 153.4 7.7 0.8 5.4 D 6.900 0.054 41.1 3.1 117.5 13.21.0 7.9 Day 8 A 7.013 0.025 29.1 1.2 110.2 10.3 7.3 0.6 B 7.004 0.00631.3 0.9 91.3 4.1 7.6 0.2 Day 9 A 6.921 0.027 30.1 1.7 107.4 4.5 6.1 0.7B 6.935 0.007 31.4 0.8 90.0 4.2 6.5 0.2Values given are the mean±SD of 4 concentrates. Days indicate the numberof days after blood collection (day 0).

During storage in the large containers the platelet concentration showedonly minimal variation during storage, whereas for the small containersabout 5% decrease was found at day 7 (Table 3b).

TABLE 7 Platelet concentration during storage bag type day 1 day 3 day 4day 7 day 8 Mean A 1172 1215 1221 1219 1233 SD 58.0 68.9 55.1 67.1 79.3Mean B 1202 1226 1250 1182 SD 69.1 65.5 64.9 46.0 Mean C 1163 SD 109.0Mean D 1137 SD 83.3Values given are the mean±SD of 4 concentrates. Days indicate the numberof days after blood collection (day 0).

D. Changes in Metabolic Parameters During Storage.

The glucose concentration measured after 1 day of storage (day 2 of PCshelf-life) was similar as observed in most other studies with storagein plasma.

Glucose consumption during storage was also similar as previouslyobserved (about 6 mM in 5 days) with a concomitant increase of lactateof about 10 mM, indicating a major role for aerobic glycolysis inglucose consumption. Hardly any differences were found between thetested types of large containers. The values for the small containerswere also very similar, but the results indicated slightly more glucoseconsumption in the small containers compared to the large containers.

TABLE 8 Glucose and lactate in supernatants of PC's during storage Day 12 3 4 7 8 9 Bag A glucose 20.7 20.0 18.8 17.3 14.1 13.7 10.4 lactate D6.6 7.9 10.1 10.2 17.0 18.1 19.1 Bag B glucose 20.7 20.3 19.2 17.7 14.314.2 11.0 lactate D 6.6 8.3 10.4 10.2 16.3 17.3 17.4 Bag C glucose 20.712.1 lactate D 6.6 21.3 Bag D glucose 20.7 13.1 lactate D 6.6 18.3Values given are the mean±SD of 4 concentrates, paired study.

The metabolic state of platelets is well characterized by theintracellular amounts of nucleotides on the various days. A correlationbetween ATP content and in vitro survival was found by Holme et al.Platelets contain two pools of nucleotides, a metabolic pool with anATP:ADP ratio of about 10, and a storage pool (in the dense granules)with an ATP:ADP ratio of about 1, resulting in an overall ratio of about2 in a total platelet extract. It was found in earlier studies thatduring storage the storage pool is depleted in favor of the metabolicpool, reflected by a slightly increased ATP/ADP ratio.

The results for the nucleotide analyses (Table 9) showed no differencesbetween the two tested containers and overall the results are similar toresults found with other studies using the B-type container at SR-BTT.The results for the small containers at day 7 (not shown) were verysimilar to the data found at day 7 for the large containers.

TABLE 9 Nucleotide content in PC's during storage units day 4 day 7 day9 A-series ATP pmol/10⁶ 51.4 ± 1.22 46.4 ± 4.83 41.9 ± 1.72 ADP pmol/10⁶27.7 ± 1.00 22.7 ± 2.25 19.2 ± 0.86 AMP pmol/10⁶ 4.3 ± 0.31 3.1 ± 0.452.8 ± 0.20 ATP:ADP 1.85 ± 0.02 2.04 ± 0.01 2.19 ± 0.09 B-series ATPpmol/10⁶ 51.2 ± 4.04 45.1 ± 1.37 42.1 ± 2.00 ADP pmol/10⁶ 27.4 ± 2.6121.4 ± 1.04 18.4 ± 0.86 AMP pmol/10⁶ 3.5 ± 0.32 3.6 ± 1.20 2.6 ± 0.24ATP:ADP 1.87 ± 0.03 2.11 ± 0.06 2.28 ± 0.07Values given are the mean±SD of 4 concentrates, paired study.

The mitochondrial membrane potential (ΔΨ) was measured with thefluorescent dye JC-1. This dye accumulates in the mitochondrial matrixin response to the membrane potential across the mitochondrial innermembrane and, above a critical concentration, J-aggregates are formedwhich show a red shift in emission characteristics as compared to themonomeric state. Therefore, the ratio of red (FL2) over green (FL1)fluorescence as measured in the flow cytometer is an indicator for theΔΨ, with a high value indicating active mitochondria and a lower valueindicative for mitochondrial damage. Table 10 shows that the FL2/FL1ratio starts high on day 2 and remains high during storage, with onlyminimal differences between the two types of containers tested.

E. Change in Membrane Characteristics of the Platelets.

Changes in cell surface antigens are indicative for the degree ofactivation of platelets during storage of PC's at room temperature.These so-called activation antigens can be detected with monoclonalantibodies (MAb). In this study we used an antibody against CD62P. Inresting platelets, the CD62P antigen is only present in the membranes ofα-granules, whereas after activation this antigen is also expressed onthe cell membrane.

Results for Large Containers

Table 10 shows the changes in expression (percentage positive cells) forCD62P from day 2 to day 9 of the PC shelf-life. The results show amoderate degree of activation, as is observed in all studies on plateletstorage, whereas there are minimal differences between the two types oflarge containers tested.

TABLE 10 Changes in cell surface markers and JC-1 during storage of PC'sCD62P AnnexinV JC-1 Series mean % SD mean % SD mean ratio SD Day 2 B 7.61.0 nd Nd Day 4 A 11.2 1.4 7.3 0.9 2.9 0.5 Day 4 B 10.9 1.4 6.6 0.9 3.00.3 Day 7 A 13.2 1.4 14.2 1.6 3.2 0.3 Day 7 B 12.5 1.9 14.8 1.2 3.0 0.2Day 9 A 19.9 2.0 16.7 2.2 3.0 0.2 Day 9 B 16.7 1.7 17.3 1.6 2.8 0.2Values given are the mean±SD of 4 concentrates (paired study); %:percentage positive platelets.

In addition to the measurement of the cell surface expression of theCD62P antigen, the percentage of cells expressing phosphatidyl-serine(PS) was measured. Under normal conditions platelets exhibit anasymmetric distribution of phospholipids in the membrane. Thecholine-containing phospholipids, phosphatidyl choline andsphingomyelin, predominantly reside in the outer leaflet, while theaminophospholipids, phosphatidyl-ethanolamine (PE) and PS, are foundmainly in the inner leaflet. PS-exposure can be measured with Annexin-Vand a high expression is generally thought to be a signal for removal ofcells from the circulation. Table 6a shows the percentage of plateletspositive for Annexin-V binding on the various days during storage,indicating that initially a very low percentage of cells are positivefor PS-exposure. During storage some increase is found, but the meanvalue at day 9 is still low, indicating a good in vitro quality.Moreover, the values found for the two types of container tested arevery similar in the paired study.

Results for Small Containers

Table 11 shows the changes in expression (percentage positive cells) forCD62P from day 2 to day 7 of the PC shelf-life for the two types ofsmall containers tested. The results show a moderate degree ofactivation, slightly higher than that observed on day 7 in the largecontainers. Only minimal differences were seen between the smallPVC-citrate bag with a representative probe (type C) and the smallPVC-DEHP bag (type D). The JC-1 ratio on day 7 was similar to that foundin the large containers.

TABLE 11 Changes in cell surface markers and JC-1 during storage of PC'sCD62P AnnexinV JC-1 Series mean % SD Mean % SD mean ratio SD Day 2 B11.6 14.4 Nd nd Day 7 C 17.6 1.5 16.8 1.6 3.2 0.3 Day 7 D 18.5 1.2 16.33.8 3.6 0.5Mean±SD for n=4, paired study.

F. Sterility at the of the Storage Period.

At the end of the storage period of 14 days (after day 9 somenon-invasive fluorescent measurements were performed, separatelyreported), one PC (2A) was found to be positive after direct seeding insoybean-casein hydrolysate or fluid thioglycolate as substrate andincubation at 20-25° C. respectively 30-32° C., according to CLBprotocol M 139 (title “Onderzoek op steriliteit”). Upon looking back tothe individual data there was no reason to exclude 2A from the data set,most probably this was a contamination introduced in the final days ofstorage, due to the frequent sampling for blood gasses (normally thesample site coupler is used 3-4 times during a study, this study up to10 times).

Conclusions

The platelet concentrates met the requirements of the research protocoland those of the European guidelines.

During storage under standard blood bank conditions, only minimaldifferences were found between the storage in the representativecontainer compared to the control Fresenius container (both PVC-citrate,paired study with full-scale platelet concentrates in plasma).

During storage under standard blood bank conditions, the variousmeasured in vitro parameters for both types of large containers tested,were similar to values observed in earlier platelet storage studies.

The values for the in vitro quality parameters at the end of study (8days of storage, 9 days of shelf-life) predicted a good in vivo recoveryand survival at the end of maximal (7 days) shelf-life.

Overall, the results of the present study indicate no harmful effectinduced by the tested representative platelet storage containers withintegrated pH probe.

The analysis of in vitro parameters on day 6 of storage in the smallcontainers indicated that the storage conditions for the down-scaledapproach were slightly worse than in the full-scale study. Therefore, itwould be better to reduce the stored volume in the small containers to13-14 mL, in order to mimic the full-scale conditions more closely.

Example 7

Bacterial Strains.

The data in FIGS. 9 and 10 were obtained by measuring various parametersof PC samples inoculated with bacteria. Bacterial strains werecultivated from −80° C. frozen stocks by two consecutive overnightpassages at 37° C. on 5% blood agar (PML, Wilsonville, Oreg.). Allstrains used were received as Microbiologics Kwik-Stiks purchased fromPML except the Escherichia coli strain (CFT073) which was received fromDr. S. Mosely at the University of Washington, Seattle, Wash. TheAmerican Type Culture Collection strains used are Staphylococcus aureusATCC 29213, Klebsiella oxytoca ATCC 43863, K. pneumoniae ATCC 13882,Serratia marcescens ATCC 43861 and Pseudomonas aeruginosa ATCC 27853.All strains except the E. coli were conditioned in plasma over threeserial passages with incubation at 22° C. than frozen at −80° C.

Processing and Inoculation of Bacterial Strains.

The small platelet storage bag depicted in FIG. 2 was used for the bagtracking studies. A sampling port (Baxter Healthcare Corp., Deerfield,Ill.) is placed in each bag. Using a 20 mL syringe (Becton Dickinson((BD)), Franklin Lakes, N.J.) and a 22G needle (BD), approximately 13-14mL of platelet concentrate (PC) is removed from the apheresis unitreceived from Puget Sound Blood Center and aseptically transferred tothe small bag for use as a normal or spiked sample. Underfilled sampleswere prepared with only 7 mL of PC. The inoculum for the spiked bags isobtained from an overnight subculture on blood agar of the desiredstrain at 37° C. without CO₂. A suspension of bacteria in sterile salineis adjusted to the turbidity of a 0.5 McFarland Standard which isroughly equivalent to 1E8 CFU/mL. This suspension is further diluted insterile saline and spiked into the prepared bags to give a finalconcentration of approximately 10, 100 or 1000 CFU/mL.

Sampling for Analysis by Bayer Blood Gas Analyzer and One-Touch UltraGlucose Meter.

Following transfer of PC and spiking of bacteria, small bags were placedin the Helmer incubator/shaker at 22° C. for 4 hours of equilibration.After initial equilibration the bags were removed, the sampling port wascleansed with 95% ethanol and a sample was removed using a 1 mL syringe(BD) with 22G needle. The sample was divided between culture for CFU/mL,glucose meter and blood gas analyzer (Bayer RapidLab model number 348.Bacterial spiked bags required a sample of ˜0.4 mL for testing. Bagspresumed to be sterile (normal or underfilled) required a sample of˜0.25 mL for testing as sterility checks were done instead of CFU/mLdeterminations.

CFU/mL determinations were performed by serial dilution (1 in 10) of 0.1mL platelets. The amount plated on Tryptic Soy Agar (PML) was 0.1 mLplatelets (and/or dilutions thereof), utilizing ˜0.2 mL of sample.Plates were incubated at 37° C., colonies were enumerated after 24 hoursand CFU/mL were calculated. The limit of quantitation is 10 CFU/mL. Onedrop of PC on sheep blood agar was used as a sterility check with theplates incubated at 37° C. for at least 4 days.

Approximately 0.15 mL of the sample was used for pH blood gas analysis(Bayer 348, Bayer HealthCare, Norwood, Mass.). Samples were runimmediately after dispensing the amount necessary for CFU/mL orsterility determinations. Readings for pH, pCO₂ and pO₂ were obtained.

Glucose was determined utilizing the OneTouch Ultra meter and teststrips (Lifescan, Milpitas, Calif.) following manufacturer'sinstructions. Sample utilized per reading was less than 10 μl andresults were recorded in mg/dL.

Example 8 Fluorescence and pH Properties of Representative SNAFLAnalogs: pKa Determination

Instrumentation.

Fluorescence versus pH of various SNAFL free acids were compared usingan Ocean Optics USB2000 fiber optic spectrometer and a tungsten halogenlight source (part number HL-2000 FHSA). The light source was equippedwith a linear variable filter that allowed the wavelength and shape ofthe excitation beam to be adjusted. The excitation wavelength wasadjusted by using a blank cuvette to the absorbance max of thefluorophore (see Table 1). A cuvette holder (part number CUV-FL-DA) wasdirectly attached to the light source and a fiber optic cable directedemitted light to the spectrometer. Excitation conditions are reportedfor each fluorescence spectrum (3000 msec irradiation at the indicatedwavelength). Spectral data were collected on a personal computer usingthe Ocean Optics software and overlays of different spectra werecaptured.

Sample Preparation.

SNAFL-1 was purchased as the free carboxylic acid from Molecular Probesin a 1 mg vial. 0.3 mL of isopropyl alcohol and 0.7 mL of water wasadded to make a 1 mg/mL solution. A molecular weight (MW) of 426 forSNAFL-1 was used to calculate molarity (SNAFL-1=2.35 mM). 4.25 uL ofthis solution was diluted to 1 mL with various 50 mM phosphate buffersto give 10 micromolar solutions with pH 6-10. 10 micromolar solutions ofSNAFL-2 (MW=460) were prepared in a similar fashion. EBIO-1 (MW=523),EBIO-2 (MW=627), and EBIO-3 (MW=489) were obtained as bulk compoundsfrom Epoch Biosciences. 1.6 mg of each solid powder was carefullyweighed out and dissolved in 3.2 mL of 40% isopropyl alcohol to give 0.5mg/mL solutions. Emission spectra were obtained for the various SNAFLand EBIO compounds at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 8.0 and 10.0.Examples of overlayed fluorescence emission spectra are shown in FIG. 26(SNAFL-1) and FIG. 27 (EBIO-3). All spectra showed an isosbesticwavelength where all emission spectra overlap (See Table 1). This is acharacteristic of ideal ratiometric performance with no competingfluorescent structures other than those shown above (lactone, naphthol,naphtholate).

pKa Calculations.

The pH at which two molecular species (tautomers) are equallyrepresented is defined as the pKa. There are many variables that canaffect pKa and methods for measurement are difficult since thestructures have overlapping absorbance. Therefore direct comparisonsfrom the literature can vary slightly. The calculations contained hereinare based on the assumption that, at pH 10, only the trianionicnaphtholate structure is present. The intensity of fluorescence at theemission maxima is divided by 2, and pH of the intersecting pH curve iscalculated by interpolation between the nearest 2 curves. The pKa of the2-chloro substituted EBIO compounds is significantly lower than theother analogs as shown in Table 1.

Example 9 The Preparation of Representative Fluorophore-ProteinConjugates: SNAFL-1/HSA

Human serum albumin (HSA) was purchased from Sigma (catalog # A-8763) as100 mg of lyophilized powder. SNAFL-1 NHS ester was purchased fromMolecular Probes as a mixture of the 5 and 6 isomers. A solution of 10mg (0.15 micromoles) of HSA in 1 mL of pH 8.56 sodium bicarbonate (0.1M) was prepared. A solution of 1 mL (1.91 micromoles) of the NHS esterin 0.1 mL of dimethylsulfoxide was prepared. 0.3 mL aliquots of the HSAsolution were transferred to a 1.6 mL Eppendorf tubes and variousoffering ratios of the NHS ester solution were added: tube 1, 11.8microliters (5 equivalents); tube 2, 23.6 microliters (10 equivalents),tube 3, 47.1 microliters (20 equivalents). The deep red solutions werevortexed and allowed to stand in the dark for at least one hour. The 5:1conjugate from tube 1 was purified by gel filtration chromatography on a0.5×20 cm column packed with Sephadex G-15 and pH 7.4 phosphate bufferedsaline (PBS). The conjugate was isolated as a fast moving red/orangeband in PBS and diluted to 0.75 mL with PBS to give a 4 mg/mL solutionof the protein conjugate. Most of the color eluted with the conjugate,but some small molecular weight (orange) impurities remained on top ofthe column. The column was clean enough to be re-used for purificationof the 10:1 and 20:1 conjugates. Each was eluted in PBS and diluted to0.75 mL to give ˜4 mg/mL solutions (60 micromolar based on HSAcomponent). The red solutions were stored refrigerated and protectedfrom light. 1 micromolar solutions of each SNAFL-1/HSA conjugate wereprepared and analyzed by UV-vis spectra using a Beckman DU640Bspectrometer. Each spectrum showed absorbance maxima at 490 and 521 nmat pH 7 as expected for the acid form of SNAFL-1 conjugates. Therelative absorbance showed the expected change in absorbance withdifferent SNAFL:HSA offering ratio. A 10 micromolar solution of SNAFL-1acid (obtained from Molecular Probes) at pH 7 was used as a standard tomore accurately determine the average loading of SNAFL-1 per each HSAconjugate preparation. Using this assay, the 5:1 conjugate had 4.1fluors/HSA, the 10:1 conjugate had 6.4 fluors/HSA, and the 20:1conjugate had 11.2 fluors/HSA.

Example 10 The Fluorescent Properties of RepresentativeFluorophore-Protein Conjugates: SNAFL-1/HSA

Relative fluorescence of various SNAFL-1/HSA conjugates and SNAFL-1 freeacid were compared using an Ocean Optics USB2000 fiber opticspectrometer and a tungsten halogen light source (part number HL-2000FHSA). The light source was equipped with a linear variable filter thatallowed the wavelength and shape of the excitation beam to be adjusted.A cuvette holder (part number CUV-FL-DA) was directly attached to thelight source and a fiber optic cable directed emitted light to thespectrometer. Excitation conditions are reported for each fluorescencespectrum (3000 msec irradiation at the indicated wavelength). Spectraldata were collected on a personal computer using the Ocean Opticssoftware and overlays of different spectra were captured. A comparisonof various loading levels of SNAFL-1/HSA showed that 4.1 to 1.6 SNAFL-1molecules gave about the same fluorescent signal. Higher loading orlower loading conjugates gave lower signals.

Emission spectra were obtained for 10 micromolar solutions in potassiumphosphate buffer. Excitation was at 540 nm. Emission maximum at 620 nmwas observed for the base form of SNAFL-1 (pH 10). As expected,intensity of 620 nm fluorescence decreased as pH decreased. Anisosbestic point at 585 nm, where fluorescence remained constant at allpH, was observed. Response was good at about pH 8, but poor between pH6-7.

Spectra obtained for a 2.5 micromolar solution of a representativeSNAFL-1/HSA conjugate (1.6 SNAFL-1/HSA) showed improved pH response forpH 6-7 (see FIG. 29). The Ocean Optics halogen light source was equippedwith a 532 nm interference filter (Edmund Optics, Barrington, N.J.) andthis allowed the fluorescent isosbestic point at 572 nm for pH 6-7 to bedetected. Emission maximum at 620 nm was observed for the base form ofSNAFL-1 (see pH 10 curve). As expected, intensity of 620 nm fluorescencedecreased as pH decreased. In comparison to the free SNAFL-1 carboxylicacid (see FIG. 26) improved response for pH 6-7 was observed for the HSAconjugate. A red shift of the pH 8 and 10 curves from the isosbesticwavelength was observed, indicative of other competing molecularstructures involving the fluorescent species. This non-ideal behaviormay be eliminated by use of a longer linker structure or a morehydrophilic linker structure between the fluorescent dye and the HSAspacer.

Example 11 The Fluorescent Properties of RepresentativeFluorophore-Protein Conjugates: EBIO-3/HSA

Fluorescence spectra were obtained for 2.5 micromolar solutions of thetwo EBIO-3/HSA conjugates prepared as described in Example 1 (Method A).The conjugates showed improved pH response for pH 6-7 (see FIG. 12 foroverlayed spectra for the 1.92:1 EBIO-3/HSA conjugate). The Ocean Opticshalogen light source was equipped with a 532 nm bandpass filter (EdmundOptics, Barrington N.J.) and this allowed the fluorescent isosbesticpoint at ˜565 nm for pH 6-7 to be detected. Emission maximum at 605 nmwas observed for the base form of SNAFL-1 (red trace, pH 10). Asexpected, intensity of 605 nm fluorescence decreased as pH decreased. Incomparison to the SNAFL-1/HSA conjugate (see FIG. 29) improved responsefor pH 6-7 was observed for the EBIO-3/HSA conjugate. A red shift of thepH 8 and 10 curves from the isosbestic wavelength was observed,indicative of other competing molecular structures involving thefluorescent species, but was of smaller magnitude than for theSNAFL-1/HSA conjugate.

Example 12 Immobilization of Representative Fluorophore-ProteinConjugates: SNAFL-1/HSA

Fluorophore-protein conjugates and fluorophore-carbohydrate conjugateswere immobilized on either nitrocellulose or capillary pore membranesusing the following general method. Fluorescein labeled dextrans with“fixable” lysine residues were obtained from Molecular Probes. Thesedextrans had a molecular weight of about 10,000 1.8 fluorophores perconjugate, and 2.2 lysines per conjugate and are sold under the tradename “Fluoro-Emerald.” Fluorescein labeled bovine serum albumin (BSA)was also obtained from Molecular Probes and had 4.5 fluors perconjugate. Various SNAFL-1/HSA conjugates were prepared as described inExample 13. Nitrocellulose membranes were obtained from Schleicher andSchuell under the trade name PROTRAN. Pore diameter was reported as 0.2microns. Capillary pore membranes made from polyester films wereobtained from Oxyphen in a variety of pore sizes. 0.1 micron and 1.0micron pore size membranes were successfully used to immobilizefluorescein dextrans. Fluorescein/dextran, fluorescein/BSA andSNAFL-1/HSA conjugates were all successfully immobilized and thefluorescent properties of the SNAFL-1/HSA conjugates were fullycharacterized as described as follows.

General Immobilization Method.

SNAFL-1/HSA (2.5 SNAFL-1/HSA) on 0.1 micron pore diameter OxyphenMembrane Discs. Fluorescent HSA conjugates with a 2.5:1 SNAFL-1:HSAoffering ratio were prepared as described in Example 4 and diluted toprovide concentrations of 0.05, 0.2, 1.0 and 4 mg/mL in phosphatebuffered saline (PBS) (pH 7.4). 5 microliter drops were applied via a 20microliter pipettor to the center of pre-punched porous discs (¼ inchdiameter) that were laid on a bench top. The spotted discs were allowedto air dry (about 30 minutes) and then placed in separate desiccatorsovernight. The dried discs were washed in separate Eppendorf tubes with2×1 mL of PBS and allowed to soak overnight in 1 mL of PBS. The washeddiscs were stable in PBS solution (no degradation after 30 days).Alternatively the discs could be re-dried in desiccators and stored dry.The wet or dry stored discs had comparable fluorescent properties. Thediscs had fluorescent signals that were proportional to theconcentration of labeled macromolecule applied to each one as measuredby the fluorescence assay described in Example 9.

Example 13 The Fluorescent Properties of Representative ImmobilizedFluorophore-Protein Conjugates: SNAFL-1/HSA

Microwell Assay of Fluorescent Macromolecular Conjugates on PorousMembrane Discs Using a Fiber Optic Spectrometer.

Fluorescent discs prepared as described in Example 8 were examined forfluorescent properties using the Ocean Optics fiber optic spectrometerdescribed in Example 14. The cuvette on the light source was replaced bya fiber optic reflectance probe which had 6 excitation fibers wrappedaround a single fiber that picks up the emitted light from the sampleand sends it to the spectrometer. The reflectance probe was threadedthrough a hole in a 12×12×18 inch black box with a lid on the front. Theprobe was clamped inside under a 1 cm square opening that allowed thetip of the probe to be positioned under a 96-well micro well plate(clear bottom black plate). The probe was tilted at a 30 degree angle toreduce reflected light entering the probe tip. The fluorescent disc ofinterest was placed in the bottom of a well and covered with 300microliters of the analyte solution of interest. The excitation lightsource was turned on long enough to position the disc of interest overthe tip of the reflectance probe, then the shutter was closed and theplate was covered with another box to shield the disc from ambientlight. Unless otherwise mentioned, the Ocean Optics software was set tocollect data with a 3000 msec integration time and 3 averages. A darkspectrum was captured with the shutter closed and used for allbackground subtracted readings during the assay. The shutter was thenopened and fluorescent reading of the disc was started. The graphicaldisplay on the computer screen gave real-time spectra after each 3000msec integration time. After the required 3 spectra were obtained (about10 seconds) the graphical display showed only subtle changes. At thispoint a snapshot of the displayed spectrum was captured and saved todisc for future processing. The shutter was closed, and the nextmicrowell experiment was set up. The same disc could be measuredmultiple times by exchanging the analyte solution in the microwells.Alternatively, different discs in different wells could be measured byre-positioning the microwell plate over the reflectance probe.

Fluorescent Loading of SNAFL-1/HSA Immobilized on Oxyphen Discs.

The microwell assay described above was used to compare therelative-fluorescence of SNAFL-1/HSA on Oxyphen discs. The excitationfilters in the halogen light source were set to a wavelength of 532 nmand a “wide open” bandpass position to maximize sensitivity of theassay. Reflectance of the excitation beam back into the detector fiberwas significant, and the wavelength position of the filter was adjustedto provide a “minimum” at 620 nm where the fluorescence from the baseform of SNAFL-1/HSA is greatest. The various concentrations ofSNAFL-1/HSA described in Example 16 were examined in separate microwellsin pH 7 potassium phosphate buffer (50 mM) as described above. Thespectra showed the ability to distinguish relative fluorescenceintensity of 4, 1, 0.2 and 0.05 mg/mL membranes at pH 7. All had signalgreater than background.

Relative Fluorescence Intensity of Various Amounts of SNAFL-1/HSA(2.5:1) Immobilized on Porous Oxyphen Discs at pH 7.

The fluorescent intensity was measured at 620 nm, the fluorescentmaximum of the base form of the fluorophore. Excitation used a wide opensetting on the halogen lamp that efficiently excites both acid and baseforms of SNAFL-1. The reflected light from the source (unmodified disc)had the lowest intensity spectrum. The spectrum of the 0.05 mg/mL discgave a small increase in fluorescence intensity. The 0.2 mg/mL disc, 1mg/mL disc, and 4 mg/mL disc showed stepwise increases in fluorescenceintensity. The fluorescence spectra of two 30 day PBS soaked sample (1mg/mL) from a different batch of membranes were essentially the same andshowed that membrane loading was reproducible from batch to batch, andthat the SNAFL-1/HSA conjugates did not dissociate significantly fromthe disc surface in PBS solution.

pH Dependent Fluorescence of SNAFL-1/HSA Immobilized on Oxyphen Discs.

The 1 mg/mL SNAFL-1/HSA discs described above were examined for pHdependent response in the microwell assay. A single disc was examined inpotassium phosphate buffers of pH 4, 5, 6, 7, 8, 9, and 10. The datashowed that these membrane discs had a wide dynamic range of pHmeasurement, but had more sensitive response at pH >6. The time betweenbuffer exchanges was 5 min, and there was no significant change inspectra after additional equilibration time. This showed that theresponse time for even dramatic changes in the pH environment of theimmobilized SNAFL-1/HSA conjugates is rapid.

“Crossover Assay” for Fluorescence Measurement of pH Using SNAFL-1/HSAOxyphen Discs.

The microwell assay described above was used to examine the fluorescentisosbestic properties of the discs. For this assay, the shutter assemblyin Ocean Optics halogen light source (part number HL-2000 FHSA) wasremoved, and two 532 nm bandpass filters (Edmund Scientific) wereinserted in the cavity using a special adaptor. This dramaticallyreduced the reflected background in the spectral region of interest(>550 nm). The data shown are for 4 mg/mL loading discs prepared withSNAFL:HSA (5:1) conjugate. The immobilized protein conjugate showedunusual pH vs. fluorescence properties in comparison to the solutionphase data. Instead of a fluorescent isosbestic point at 575 nm, therewas a stepwise increase in the fluorescent intensity as pH increased.The pH 10 spectrum showed the expected maximum at 620 nm, and crossedthe overlaid spectral curves obtained in pH 4, 6, 7 and 8 buffers. These“crossover points” were used as the basis for a sensitive assay todetermine pH of the membrane environment. Three different membrane discswere examined using this assay format on three different days. Thecrossover points were reproducible within 2 nm.

The 4 mg/mL discs (3.6:1 SNAFL-1:HSA) showed stepwise increase in pH 10“crossover.” The crossover was at 579 nm for pH 4. Three discs/threedifferent days gave the same result ±2 nm. The crossover points were at592 nm (pH 6), 600 nm (pH 7a,b), and 611 nm (pH 8). The fluorescentmaximum at pH 10 was at 620 nm, similar to the solution phaseproperties.

Example 14 The Fluorescent Properties of Representative ImmobilizedFluorophore-Protein Conjugates: Ebio-3/Hsa

Telescoping Tubing Insert Assay of Fluorescent Macromolecular Conjugateson Porous Membrane Discs Using a Fiber Optic Spectrometer.

Fluorescent discs prepared as described in Example 2 were examined torelate the fluorescent properties to the liquid phase pH using the OceanOptics fiber optic spectrometer described in Example 17 with the dual532 nm filtered (Edmund Scientific) halogen light source (part numberHL-2000 FHSA). A holder for a 5/32 inch membrane disc was crafted with 4mm OD and 5 mm OD polystyrene telescoping tubing and an angled 0.015 inthick polystyrene window. The angled window was placed so that it heldthe membrane disc at a 60 degree angle relative to the tubing axis. Thisallows the fiber optic probe to be placed in one end of the tubing andinterrogate the disc on the other side of the window which is contactwith a liquid of a certain pH. Buffers of known pH values were placed incontact with the telescoping tubing inserts and discs made by thespotting immobilization method in Example 2 and fluorescent emissionsrecorded with the Ocean Optics software set to collect data with a 1000msec integration time and 3 averages.

For liquids with unknown pH values, a stirred and light protected vesselcontaining 5 telescoping tubing inserts and discs made by the soakingimmobilization method in Example 2, 50 mL of buffer or plasma, and acalibrated pH electrode (ROSS electrode/Orion 720a meter) was used tostudy the pH and fluorescent response of the fluorescent discs. Drops of1 N HCl or 1 M NaOH were added to create a range of pHs from liquidsstudied. Fluorescent spectra were collected through Ocean Optics macrosin Excel set to read for 1000 msec integration time and three averages.The spectra were analyzed using the modeled bandpass filters andratiometric method in Excel to obtain calibration curves for PBS,platelet poor plasma and platelet rich plasma.

Injection Molded Insert PVC Tube Assay of Fluorescent MacromolecularConjugates on Porous Membrane Discs Using a Custom OptimizedFluorescence Based pH Detector.

Injection molded polycarbonate parts were fashioned to fix thefluorescent discs to the fluorescence pH detector probe as pictured inFIG. 22. Membranes were prepared as described in the soakingimmobilization method in Example 2 and assembled into the plasticinsert. A 1 in long and 3/16 in ID PVC tube was placed on the spike endof the insert such that 250 ul of liquid was placed in the tube andcovered with parafilm to slow carbon dioxide desorption. A fluorescentmeasurement of the first and second wavelengths was taken and then thepH was read by a blood gas analyzer (Bayer 348). The pH of plasmasamples were adjusted by acid and base additions as in the telescopingtubing insert assay to create the range of pH data.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method for monitoringa parameter of one or more samples positioned in an incubator,comprising: (a) irradiating a fluorescent species having a firstemission intensity at a first emission wavelength and a second emissionintensity at a second emission wavelength, wherein the first and secondemission wavelengths are not the same, each emission intensity dependenton a parameter, with excitation light emanating from a probe physicallyisolated from the fluorescent species to provide first and secondemission intensities, wherein the fluorescent species is in a samplecontained in a vessel in an incubator; (b) measuring the first andsecond emission intensities to determine a first parameter reading ofthe sample; and (c) repeating step (a) after a pre-determined time andmeasuring the first and second emission intensities to determine asecond parameter reading of the sample.
 2. The method of claim 1,wherein step (c) is repeated to provide multiple parameter readings tomonitor the parameter of the sample over time.
 3. The method of claim 1,wherein the parameter is pH.
 4. The method of claim 1, wherein theparameter is CO₂.
 5. The method of claim 1, wherein the parameter isoxygen.
 6. The method of claim 1, wherein the parameter is glucose. 7.The method of claim 1 further comprising measuring the first and secondemission intensities of each sample positioned in the incubator todetermine a first parameter reading for each of the samples.
 8. Themethod of claim 7, wherein step (c) is repeated to provide multipleparameter readings to monitor the parameter of each of the samples overtime.
 9. A method for monitoring the pH of one or more samplespositioned in an incubator, comprising: (a) irradiating a fluorescentspecies in a sample contained in a vessel with excitation lightemanating from a probe physically isolated from the fluorescent species,wherein the excitation light has a wavelength sufficient to effectfluorescent emission from the fluorescent species, wherein thefluorescent species exhibits a first emission intensity at a firstemission wavelength and a second emission intensity at a second emissionwavelength, the ratio of the first and second emission intensities beingdependent on pH, wherein the first and second emission wavelengths arenot the same; (b) measuring the first and second emission intensities todetermine the first pH of the sample in the incubator; and (c) repeatingstep (a) after a pre-determined time and measuring the first and secondemission intensities to determine the second pH of the sample in theincubator.
 10. The method of claim 9, wherein step (c) is repeated toprovide multiple pH readings to monitor the pH of the sample over time.11. The method of claim 9 further comprising measuring the first andsecond emission intensities of each sample positioned in the incubatorto determine a first parameter reading for each of the samples.
 12. Themethod of claim 9, wherein step (c) is repeated to provide multipleparameter readings to monitor the parameter of each of the samples overtime.